The phase transition of VO2 films in 90° twisted E-shaped resonators enables high-efficiency thermal modulation of linear polarization conversion. The asymmetric transmission of linearly polarized wave and circular dichroism simultaneously exhibit a pronounced switching effect dictated by temperature-controlled conductivity of VO2 inclusions. The proposed hybrid metamaterial design opens exciting possibilities to achieve dynamic modulation of terahertz waves and further develop tunable terahertz polarization devices.Polarization manipulations of electromagnetic waves can be obtained by chiral and anisotropic metamaterials routinely, but the dynamic and high-efficiency modulations of chiral properties still remain challenging at the terahertz range. Here, we theoretically demonstrate a new scheme for realizing thermal-controlled chirality using a hybrid terahertz metamaterial with embedded vanadium dioxide (VO Polarization provides a controllable degree of freedom in the field of light propagation and information processing. Polarization devices are crucial in many optical systems. As a fundamental phenomenon, optical activity, that is the ability to rotate the polarization state of light, has acquired great importance in many fields of science including physics, life-sciences and chemistry235678910112) and semiconductors enable active THz metamaterials that are excited by external stimuli via electric bias232425262829303133343536373956789101112161735363735323Sb2Te62Sb2Te52 can exhibit an insulator-to-metal phase transition that can be electrically282930314243442 is much more preferable282930314243442 transition behavior could be tailored by extreme nanogap42432 with a metamaterial has been shown a large temperature activated tuning of the transmission without polarization conversion in the THz range. It is possible and worthwhile that the asymmetric transmission of linearly polarized wave and circular dichroism can be efficiently modulated by the phase transition effect of VO2, which provides an alternative route to realize switchable and functional THz devices. Actually, some other efficient approaches have been also presented to manipulate the polarization state with metasurfaces. For instance, broadband anomalous deflection can be observed with phase discontinuities47Terahertz (THz) wave occupies a large portion of the electromagnetic spectrum between microwave and infrared frequencies. A variety of metamaterials have been proposed to offer a strong engineered THz response and fill the so-called THz gap131415161718192021161920212 phase transition. The hybrid THz metamaterial is comprised of an array of 90°-twisted E-shaped resonators with incorporated VO2 films. The orthogonal arrangement leads to a strong asymmetric transmission and circular dichroism in the bilayered metamaterial. The insulator-to-metal transition in VO2 films changes the resonator structure as well as the resonant frequency. The hybrid chiral metamaterial allows us to dynamically modify its chiral properties by controlling the temperature-dependent conductivity of VO2. Therefore, the thermal switching of the asymmetric transmission and circular dichroism can be implemented. Such dynamic control of phase transition metamaterials is of importance to acquire a variety of functionalities in the THz regime, such as filters, modulators and switches.In this work, we report a novel metamaterial-based thermal switch to control artificial chirality and THz wave propagation exploiting the mechanism of VO2 inclusions. The period of the unit cell is d = 100 μm. Each dimer is composed of two spatially separated E-shaped gold resonators with a thickness of tm = 200 nm. The two E-shaped resonators are geometrically identical, but the back layer is twisted by θ = 90° along the z axis with respect to the front one. The spacer dielectric layer is polyimide with a thickness of t = 16 μm. The E shape is square with a length of a = 80 μm. The width of the metallic strip is w = 15 μm. In order to realize a tunable THz metamaterial, VO2 films with a size of b = 17.5 μm and l = 15 μm are incorporated into one of the gaps of E-shaped resonators. The thickness of VO2 films is identical to that of metal layers, i.e., 200nm. The displacement of VO2 is described by s. VO2 film layer can be deposited on the polyimide dielectric by the reactive magnetron sputtering technique2 islands and E-shaped metallic resonators can be fabricated by CF4/O2 plasma etching and the photolithography technique282 film integrated into the chiral metamaterial will undergo an insulator-metal phase transition process, accompanied by conductivity change up to several orders of magnitude2 films has relatively simple geometry, easy integration and more flexibly switching function for the cross-polarization transmission. Besides, a flat cross-polarization transmission and asymmetric transmission can be easily achieved with respect to other simple metamaterial structures. In addition, switching functionality based on phase transition property of VO2 can be applied to other metamaterial designs in microwave, THz and near-infrared frequencies28293050The proposed metamaterial configuration is sketched in z direction, Jones matrix is used to explore the polarization properties of the hybrid metamaterial. Jones matrix links the complex amplitudes of the incident to the transmitted field:For a linearly polarized wave beam normally incident on the structure along the -Ei and Et denote the incident and transmitted electric field, the matrix tf represents the transmission matrix along forward propagation. The matrix tf consists of 2 × 2 elements, in which tjk and |tjk| indicate the transmission complex coefficients and amplitudes. The subscripts j and k correspond to the polarization states of the transmitted and the incident waves, which could be either x or y linear polarization. Here, the two layers are structurally identical, while the second layer structure is twisted clockwise by 90° around the z axis. So, txx is equal to tyy. Since the reciprocal theorem is applied, the transmission matrix tb for backward propagation  can be derived as9where the vectors In order to further quantify the polarization properties of the metamaterial, the circular transmission coefficients can be inferred from the linearly polarized transmission matrix according to the following equation:where “+” and “−” denote the right-handed (RCP) and left-handed circularly polarized waves (LCP), respectively.εp = 2.4 + 0.005i Au = 4.561 × 107 S/m. Generally, the complex dielectric properties of VO2 films in the THz range can be described by the Bruggeman effective-medium theory (EMT)Numerical simulations were performed in the THz range by means of the commercial software CST Microwave Studio, where the lossy polyimide spacer was assumed to have a relative permittivity εd and εm, respectively, are dielectric constants of the insulating and metallic phase VO2 films, and V is the volume fraction of the metallic regions. For simplicity, the temperature-dependent conductivity 2 films28292 films in the insulating state is about 9, while the conductivity in the insulating state is smaller than 200 S/m and as high as an order of 105 S/m in the metallic statewhere 2 films embedded into the bilayered E-shaped chiral metamaterial experience an insulator-to-metal phase transition under thermal excitation, further modulate the polarization conversion and asymmetric transmission due to the structure change of resonant elements. The electromagnetic responses of the hybrid metamaterials with VO2 films at different temperatures were modeled using the conductivity 2 conductivity from 200 to 105 S/m corresponds to the insulator-to-metal phase transition2829302 conductivity reaches the value of the metallic stateVO2 films for forwardly propagating electromagnetic waves along −z direction, where the displacement s = 0. In 2 film is in the insulating state with txy| = 77%. Interestingly, this dispersion-free property of the cross-polarization transmitted light only occurs at a specific dielectric layer thickness. The dispersion-free property can be well engineered by the metamaterial’s parameters56572 could actually be cancelled out by the thickness–dependent dispersion of the dielectric layer2 film undergoes a change from the insulator phase to the metal phase accompanied by an increasing conductivity. Accordingly, the y-to-x cross-polarization transmission has an obvious reduce. The complete insulator-to-metal phase transition of the metamaterial makes the y-to-x cross-polarization conversion into the “OFF” state. When 5 S/m, corresponding to the temperature of about 85 °C, the y-to-x cross-polarization conversion |txy| almost decreases less than 0.1 at about 1.4 THz. Particularly, the modulation depth of the y-to-x cross-polarization transmittance is as high as MOD = 96.6%, calculated by tyy and txxdepend weakly on tyx is totally suppressed below 0.1 in the frequency range of 1.25–1.50 THz no matter what the conductivity 2 film in the front layer are shown in 2 film integrated metamaterial is available. However, the modulation depth of the y-to-x cross-polarization transmittance decreases to 76% due to slightly high transmission in the metallic state of VO2 film, here the metamaterial can be regards as a combination of E-shaped and 6-shaped resonators. Therefore, the hybrid chiral metamaterial incorporated with single-layer VO2 film can alternatively realize a thermal switching effect of the cross-polarization transmission.On the contrary, no pronounced switching effect occurs for three other transmission coefficients. The co-polarization transmission 2 films. To visualize the thermal controlled polarization effect, the polarization rotation angle θ and its ellipticity angle η of the transmitted wave are calculated according to Next, we mainly focus on the chiral switching response of the hybrid metamaterial with bilayered VOθ and its ellipticity angle η. When VO2 film is insulating, at about 1.42 THz the transmitted wave is linearly polarized since the polarization rotation angle nearly approaches 60° and the ellipticity angle is close to 0, while at about 1.37 THz the transmitted wave is circularly polarized due to η = 45°. But VO2 film is metallic with its conductivity 5 S/m, both the polarization rotation angle θ and its ellipticity angle η are almost 0, thus its chirality is thermally switched off. The giant chirality modification results from the change of the effective metallic geometry when VO2 film experiences a phase transition. Importantly, the hybrid metamaterial can realize thermally controlled linear-to-circular polarization conversion at around 1.37 THz.txy| ≠ |tyx| and |txx| = |tyy|, the hybrid metamaterial will reveal the asymmetric transmission of linearly polarized wave. Asymmetric transmission for the linearly polarized wave refers to total intensity difference between two opposite directions and the associated asymmetric transmission parameter can be expressed as:Since |2 films enables this hybrid metamaterial to efficiently switch the asymmetric transmission of linearly polarized waves as shown in x and Δy are exactly contrary to each other. Remarkably, the asymmetric transmission parameter can be thermally modulated between 0.6 and 0 in the frequency band of 50 GHz centered at 1.40 THz. Generally, the metamaterial exhibiting asymmetric transmission of linearly polarized wave is anisotropic and 3D chiral. Thus, the hybrid VO2 metamaterial can also thermally control its circular dichroism. The circular dichroism refers to differential transmittance between RCP and LCP waves, which can be calculated by CD = |t++|2 − |t−−|2. Interestingly, the circular dichroism strongly depends on the conductivity of the VO2 film shown in 2 film leads to a complete switching effect of the circular dichroism with a maximum change of 0.45 at around 1.36 THz. Our demonstration could be of importance to modulate THz wave propagation and recognize interaction between the external stimuli and matter by detecting its chiral properties. The scheme could be exploited for developing novel highly efficient THz polarization modulators and thermal sensors.The insulator-to-metal phase transition of VO2 metamaterial at 1.4 THz are simulated and presented in 2 film has a very low conductivity and the resonator is E-shaped. The strong antiphase current pairs excited by incident y-polarized wave can result in a magnetic response between the two layers shown in H2 and the incident electric field E. Therefore, the strong chirality can be observed. While with thermal excitation, VO2 film behaves as metal material and the resonator is 6-shaped. In this case, no strong antiphase current pairs and magnetic response are excited in 2.To clarify the physical origin of thermal-controlled switching polarization effect, the Born-Kuhn model can be recalled, in which two charged oscillators couple with each other22 film, shown in 2 conductivity. In addition, the hybrid metamaterial has the largest switching modulation depth as the thickness of polyimide is about 17 μm. When the displacement s is less than 10 μm and the length l of VO2 films is larger than 10um, the thermal switching effect can be observed in In order to know how geometrical parameters of the metamaterial affect the thermal switching effect, we also perform numerical simulations of the hybrid metamaterial, in which the only one parameter is variable and the others are kept unchanged. The dependence of the switching properties at resonant frequency 1.4 THz is individually investigated on the thickness of polyimide dielectric layer, the displacement and length of VOtxy is almost kept unchanged, but the resonant frequency shifts slightly to red. For angles ranging from 50° to 80°, the modulation depth of the cross-polarization transmission txy has a rapid damping. As a result, no thermal switching effect can be observed in the hybrid chiral metamaterial at a large angle of incidence. It is worth mentioning that the thermal switching functionality in our metamaterial remains fairly available for a wide range of angles up to 40° and thus our design is robust and flexible to manipulate THz wave propagation.In general, extrinsic 2D and 3D chirality in intrinsically non-chiral metamaterials can be dominated and enhanced by increasing angles of incidence62 films embedded in two 90°-twisted E-shaped resonators promises a dynamic control on polarization rotation angle, circular dichroism and asymmetric transmission of linearly polarized light under external thermal excitation. The transmission maximum is close to |txy| = 77% at about 1.4 THz. Particularly, our simulations show that the thermal switching functionality remains fairly available for a wide range of angles up to 40°. In addition, thermal switching effect can be engineered to work at other frequencies by changing geometrical parameters. Most importantly, the phase change material VO2 can be easily completed. Our work offers flexible polarization modulation in the THz frequency that would be highly beneficial for potential applications such as thermal switching and polarization sensitive detection.In conclusion, we have demonstrated a thermal-controlled chiral switching in a hybrid THz metamaterial and the modulation depth of 96.6% is observed at about 1.4 THz for the linear cross-polarization transmittance. The insulator-to-metal phase transition of VOεp = 2.4 + 0.005iAu = 4.561 × 107 S/m. Generally, the complex dielectric properties of VO2 films in the THz range can be described by the Bruggeman effective-medium theory (EMT)2 films28292 films in the insulating state is about 9, while the conductivity in the insulating state is smaller than 200 S/m and as high as an order of 105 S/m in the metallic stateNumerical simulations were performed in the THz range by means of the commercial software CST Microwave Studio. In the model, the hybrid metamaterial is freestanding that could be possibly fabricatedHow to cite this article: Lv, T. T. et al. Hybrid metamaterial switching for manipulating chirality based on VO2 phase transition. Sci. Rep.6, 23186; doi: 10.1038/srep23186 (2016).


Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied.in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1–110 contains two repeats formed each of two β sheets followed by a α helix (amino acids 1–58 and 58–110) that are important for the formation and stability of polyhedra. These fragments 1–58, 58–110 and 1–110 could be incorporated into polyhedra. However, only fragments 1–110 and 58–110 can self-aggregate.Based on the crystal structure of polyhedrin, we conducted an These results demonstrate that 58–110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra. The development of eukaryotic systems for the expression of recombinant proteins has been a major goal in biotechnology due to its widespread utility in medicine, veterinary medicine, and agriculture, among other related areas .Autographa californica nucleopolyhedrovirus (AcMNPV), and Bombyx mori nucleopolyhedrovirus (BmNPV). The third uses the RNA virus Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) cypovirus. In nature, the viruses of these 2 families are protected from adverse environmental conditions as they are occluded into crystalline lattices called polyhedra or occlusion bodies, derived mainly from a single viral protein called polyhedrin [The use of insect viruses to produce and to obtain different recombinant proteins has grown in recent decades ,3. Threelyhedrin . The occlyhedrin .Polyhedrin is one of the most abundant proteins in a baculovirus-infected cell, since its expression is driven by a very strong promoter . BecauseSince BmNPV and BmCPV polyhedra are particles of about 1 μM in diameter and can be easily purified by centrifugation, they represent good candidates to express recombinant proteins. Using this strategy, Je et al. incorporated the green fluorescent protein (GFP) into the AcMNPV polyhedra by fusing it to the carboxyl terminus from the polyhedrin gene . HoweverThe polyhedrins of baculoviruses and cypoviruses do not share a similar amino acid sequence . HoweverIt has already been shown that the crystal structure from both the AcMNPV and the BmCPV polyhedra is an arrangement of polyhedrin trimers, which are interconnected through their amino N-terminal helices . These iDespite the similarities in crystal symmetries and identical unit cell dimensions, structures of baculovirus and cypovirus polyhedrins are different at the atomic level. Both structures have a β-sandwich core domain, with projecting C- and N-terminal helices, but the topologies are dissimilar and the helices interact differently .Based on these findings, Ijiri et al. incorporated several foreign proteins into BmCPV polyhedra by fusing them to the first 30 amino acids of polyhedrin, which contains an α helix known as H1 . BecauseMore recently, recombinant polyhedra in BmNPV have been obtained by co-expressing the foreign proteins fused to the first 110 amino acid N-terminal fragment in combination with the complete WT polyhedrin ,17. The Because AcMNPV is one of the most widely used systems to express recombinant proteins, and given the fact that less is known about what fragments in the polyhedrin protein are sufficient to incorporate foreign proteins into polyhedra, we aimed the present study at determining the minimal fragment that can be used to incorporate foreign proteins into AcMNPV polyhedra. We first analyzed the amino terminal of AcMNPV polyhedrin considering the known crystal structure of the protein  supplemented with 10% heat-inactivated fetal bovine serum  , 1X Yeastolate , 1X Lactalbumin , and 1X Antibiotic-antimycotic  according to standard methods. For suspension cultures, pluronic acid F-68 at a final concentration of 0.1% was added and the cells were sub-cultured every 2 to 3 days. The Bac-N-blue system  was used for the construction of the recombinant baculovirus (see below). The WT and recombinant AcMNPVs used in the present study were propagated in Sf9 cells.The The complete polyhedrin gene was obtained by PCR amplification using the Baculovirus forward and reverse PCR primers  and cloned into pEGFP-C2 . The recombinant baculoviruses with the different fragments of polyhedrin were constructed by digestion with restriction enzymes or by PCR amplification of the constructions. These PCR fragments were cloned to pEGFP-N1, −N2 or -N3  as needed to obtain the fusion proteins with an open reading frame. The recombinant plasmid vectors were confirmed by restriction endonuclease analysis and sequencing. All the fusion genes of EGFP-polyhedrin were then sub-cloned into the pBlueBac4 plasmid , which was used with the Bac-N-blue transfection kit  to obtain the recombinant baculoviruses. The recombinant baculoviruses were purified and then amplified to obtain high titer virus stocks. The baculovirus titer was obtained and expressed as plaque forming units (pfu) per milliliter according to standard protocols provided by the manufacturer .2PO4, 20 nmol/L Na2HPO4, 150 mmol/L NaCl, pH 7.2) , and fragmented ultrasonically three times for 30 s each , followed by centrifugation at 12,000 g at 4°C for 10 min. The pellets were then washed 2X with PBS and then finally resuspended in PBS buffer.Sf9 cells co-infected with the recombinant and WT viruses were collected by centrifugation at 96 hrs post-infection, resuspended in phosphate buffered saline . The polyhedra or cells were washed three times with PBS and fixed with mounting medium . The infected insect cells were incubated with DAPI , Molecular Probes, USA) at a dilution 1:1000 for 5 min before fixation with DAKO Cytomation fluorescence mounting media . Images were collected with an Olympus FV1000 confocal microscope and analyzed using Fluoview 10-ASW-2.1 software . Image acquisition using transmission electron microscopy (TEM) of infected cells was conducted according to established protocols. Briefly, the cells were washed with 0.08 M cacodylate buffer  and fixed for 10 min with 0.6% glutaraldehyde  and 0.4% paraformaldehyde in 0.08 M cacodylate buffer, pH 7.4. Post-fixation was made with 1% osmium tetroxide  in cacodylate buffer Thin sections were counterstained with uranyl acetate for 10 minutes and with lead citrate for 2.5 minutes. Observations were made in a Jeol 1010 electron microscope .The polyhedra crystals containing the different fragments of polyhedrin fused to EGFP were purified as indicated above. Purified crystals were subjected to confocal microscopy and TEM to validate its purity. In all cases basically no cellular debris was observed. Polyhedra crystals were introduced into the sorting chamber of a fluorescence-activated cell sorting (FACS) apparatus . Event counting was terminated at 10,000 events as previously described . Fluores(1–110)EGFP and PH(58–110)EGFP were introduced in the system at two different concentrations. Particles sizes and particle concentration was obtained from direct measurements with NanoSight. Data represents the analysis from millions of events and is given by particle sizes per milliliter  particle analysis . This ter Figure D.Figure http://www.rcsb.org/pdb/explore.do?structureId=2wuy)) was utilized in the identification of the polyhedrin secondary structure, which directed the generation of the different polyhedrin fragments reported in this study.The crystal structure of the wild type AcMNPV polyhedrin -EGFP), and the different fragments of polyhedrin: amino acids 1–25 (PH(1–25)EGFP), 1–34 (PH(1–34)EGFP), 1–48 (PH(1–48)EGFP), 1–58 (PH(1–58)EGFP), 17–58 (PH(17–58)EGFP), 58–110 (PH(58–110)EGFP), 1–110 (PH(1–110)EGFP), and 111–245 (PH(111–245)EGFP). All recombinant genes were under the control of the polyhedrin promoter in the recombinant baculoviruses. All polyhedrin fragments were cloned at the N-terminal of the EGFP except for PH(1–245)EGFP which was cloned at both the N- and C-terminal of the EGFP. Because we obtained indistinguishable results with both constructs, we will describe here only PH(1–245)-EGFP. In order to obtain polyhedra, a second recombinant baculovirus carrying the wild type copy of polyhedrin was utilized in co-infections with all baculoviruses carrying fragments from the polyhedrin gene (listed above).In order to visualize the expression and localization of polyhedrin and polyhedra in the infected cells, we developed several recombinant baculoviruses containing different fragments from the polyhedrin gene fused to the enhanced green fluorescent protein (EGFP). The baculovirus were designated according to the amino acid fragment of polyhedrin: WT polyhedrin (PH(1–245)-EGFP was analyzed by confocal microscopy in Sf9 infected cells. Figure (1–245)-EGFP did not form polyhedra. Similar results were observed in cells infected with the same polyhedrin fragment cloned in the N-terminus of EGFP (data not shown). In both cases EGFP was observed as aggregates  localized at the cytoplasm of the cells. EGFP never co-localize with DAPI counterstained nuclei, demonstrating the cytosolic localization of PH(1–245)-EGFP -EGFP. In all these experiments two viruses were used, one carrying a WT copy of full-length polyhedrin, and a second virus carrying the corresponding polyhedrin fragment fused to EGFP. After performing a WT baculovirus- PH(1–245)-EGFP baculovirus co-infection titration, we concluded that the ratio of WT polyhedrin to recombinant PH(1–245)-EGFP was critical for the formation of polyhedra -EGFP and the WT polyhedrin, obtained by maintaining the multiplicity of infection (MOI) of the baculovirus PH(1–245)-EGFP at 1 and by increasing the MOI of baculovirus carrying the WT copy of polyhedrin from 0.5, 1, 2, 3 and 5 MOI. In Figure (1–245)-EGFP was used. This result revealed that WT polyhedrin had to be co-expressed in order for recombinant polyhedrin fragments carrying EGFP to locate and assemble into polyhedra in the nuclei of the cells. The requirement of WT polyhedrin for the formation of recombinant polyhedra has been previously demonstrated for other baculoviruses [(1–245)-EGFP. Increasing the ratio of PH(1–245)-EGFP results in soluble protein, suggesting that all possible sites have been occupied in the polyhedra and the excess PH(1–245)-EGFP is discarded from the crystal structure.In order to obtain recombinant polyhedra with EGFP incorporated into the crystals, we performed a series of co-infections using both WT and recombinant baculoviruses carrying PH1–24-EGFP. In1–24-EGFP. In1–24-EGFP. Inoviruses ,21,22. I(1–110)EGFP aggregated in the nuclei , indicating that the self-aggregation property of polyhedrin is retained in this fragment -EGFP, Figure (1–245)-EGFP and PH(1–110)EGFP retained the property of self-aggregation but the carboxyl terminus of polyhedrin (PH(110–245)EGFP) is soluble, strengthens the hypothesis that in the fragment PH(1–110)EGFP is located the sequence/structure responsible for self-aggregation.To evaluate the expression and localization of the recombinant baculoviruses containing the fused EGFP protein to different fragments of polyhedrin, we infected Sf9 cells with baculoviruses carrying the different fragments generated for this study Figure A and pert Figure B. These igure 1–25-EGFP, F(1–110)EGFP showed electron dense intranuclear protein aggregates, corresponding to the polyhedralike structures observed in confocal microscopy -EGFP recombinant polyhedrin (data not shown).TEM images from cells expressing PHy Figure C. Notice(1–110)EGFP with WT polyhedrin resulted in the formation of canonical polyhedra with EGFP contained within the crystal EGFP baculovirus, the EGFP was observed scattered throughout the cell as a soluble protein EGFP was observed as a soluble protein in the cytosol and nucleus of infected cells, but the fragment PH(58–110)EGFP was observed as condensed, amorphous material in the nuclei and cytosol of the infected cells EGFP is soluble and does not aggregate on its own. Nevertheless both fragments can be incorporated into canonical polyhedra when co-expressed with WT polyhedrin. The fragment PH(1–110)EGFP can self-aggregate, but the aggregates are contained within the nuclei, strongly suggesting that the nuclear retention signal is found in the combination of fragments PH(1–58)EGFP and PH(58–110)EGFP, since separately both fragments are distributed in the nuclei and cytosol, but when combined (in the fragment PH(1–110)EGFP) are exclusively present in the nuclei.Most interestingly, the baculoviruses expressing shorter fragment PHs Figure A. Co-exps Figure B. These These results suggest the presence of a putative nuclear retention signal shared by fragments 1–58 and 58–110. A previous study identified a nuclear retention signal present within the sequence 1–110 . HoweverTo delimit more precisely the minimum amino acid sequence capable of forming canonical polyhedra when co-expressed with WT polyhedrin, we produced several recombinant baculoviruses carrying different fragments from the amino terminus of polyhedrin. We concentrated in the first 58 amino acids, to further delimit the minimal sequence capable of being incorporated into polyhedra.(1–25)EGFP and PH(25–58)EGFP did not form aggregates, neither they could be incorporated into the polyhedra when co-expressed with WT polyhedrin. Similar results were obtained with the fragment PH(1–34)EGFP, PH(1–48)EGFP, PH(17–58)EGFP or PH(25–49)EGFP. These results indicate that the minimum fragment that can be incorporated into the polyhedra crystal is PH(1–58)EGFP. Similarly, the fragment PH(58–110)EGFP was also incorporated into the polyhedra crystal, but we did not explore other sequences within this later fragment in the present study.The fragments PHAll the results presented in this study show that: a) fragments from the n-terminus of polyhedrin, namely 1–58 and 58–110 can also be incorporated into the canonical polyhedra (when co-expressed with WT polyhedrin), regardless of the cellular localization or the fact that they can self-aggregate (58–110) or not (1–58); b) fragments from the c-terminus of polyhedrin are soluble, found scattered throughout the cell cytosol and nuclei, and are not incorporated into the polyhedra (when co-expressed with WT polyhedrin). In fact, they were completely excluded from the polyhedra crystal, as illustrated in Figure To obtain quantitative data about the effectiveness of the different fragments to be incorporated into the polyhedra crystal, we conducted flow cytometry studies with polyhedra formed by the combination of the different fragments from polyhedrin fused to EGFP and WT polyhedrin. Polyhedra were purified for these studies prior to conducting the flow cytometry studies .(1–58)EGFP, PH(58–110)EGFP, PH(1–110)EGFP and the full length PH(1–245)-EGFP were incorporated into polyhedra crystals. Most interestingly, the most fluorescent polyhedra was obtained with the fragment PH(58–110)EGFP, followed by PH(1–110)EGFP and the full length PH(1–245)-EGFP. These results strongly suggest that using the fragment PH(58–110)EGFP results in higher yields of recombinant protein incorporated into the polyhedra crystals.Figure (1–110)EGFP and PH(58–110)EGFP produced electron dense particles identifiable by TEM. Interestingly, both nanoparticles can be isolated from Sf9 cell lysates and retain its structural features. Nanoparticles produced by fragment PH(1–110)EGFP inside Sf9 infected cells are illustrated in Figure (1–110)EGFP can be isolated from Sf9 lysates, as illustrated in the electron scanning images from Figure (1–110)EGFP and PH(58–110)EGFP produced nanoparticles of about 100 nm in diameter (6D). Notice that the main nanoparticles identified are multiples of 100 nm, which was the most abundant and smallest size identified (6D).As we have previously shown, both PHFigure (58–110)EGFP as containing the sequence/structure essential for the self-aggregation properties of polyhedra EGFP and PH(58–110)EGFP) when co-expressed with WT polyhedrin have similar structural features: both are formed by two consecutive β sheets followed by a α helix EGFP retains the self-aggregation property of polyhedra.In spite of the fact that both minimal fragments that can be incorporated into polyhedra (PH–58EGFP (1–58)EGFP and PH(58–110)EGFP. For this reason the fragment PH(1–110)EGFP is also incorporated into polyhedra when co-expressed with WT polyhedrin. The fragment PH(111–245)EGFP from the C-terminal region of polyhedrin was excluded from the polyhedra crystals EGFP EGFP and PH(58–110)EGFP), but the self-aggregation property is present exclusively in fragment PH(58–110)EGFP. The sequence/structure required for nuclear localization appear to be a combination of fragments PH(1–58)EGFP and PH(58–110)EGFP, since only the fragment PH(1–110)EGFP showed exclusive nuclear localization.All the results gathered in this study indicate that the sequence/structure required for a fragment to be incorporated into the polyhedra crystal are present in both fragments PHs Figure D. Notewont PH111–45EGFP fThe baculovirus expression system has become a powerful tool for recombinant eukaryotic gene expression . The iniJarvis et al. fused different fragments of AcMNPV polyhedrin to two nonnuclear reporter proteins, β-galactosidase and β-glucorinadase to define its possible nuclear localization signal, and study the distribution and assembly of polyhedra by indirect immunofluorescence and by biochemical fractionation and SDS-PAGE of infected Sf9 . The recMore recently, Je et al. using GFP fused to AcMNPV polyhedrin, observed the GFP in a soluble form in the nucleus and cytoplasm of infected cells . Only whA, βA’, and βA”, βB ), followed by a short α helix (α1 and α2) Figure The N-terminal from 1–110 amino acids of AcMNPV polyhedrin can be divided into two similar fragments (1–57 and 58–110) each of them consisting of two β helices , PH(58–110) and PH(1–110) of AcMNPV can be incorporated into polyhedra, only when co-expressed with the WT polyhedrin. The fact that polyhedra located in the nucleus is observed only when co-expressing WT and recombinant fragments of polyhedrin, suggest that the WT and the different N-terminal fragments identified in this study associate in the cytosol and travel, assembled with WT polyhedrin, to the nucleus of the infected cell. In agreement with this hypothesis, a previous study demonstrated that the nuclear localization of polyhedrin becomes more evident during the phase of occlusion due to the high rate of polyhedrin localization signal and a higher rate of protein biosynthesis [We observed that the fragments PHynthesis .An important finding in the formation of polyhedra was the ratio of the recombinant fused polyhedrin fragments to the WT polyhedrin. Because the minimal unit of polyhedra crystals is a trimer, the association of the recombinant protein could be carried out in a stoichiometric ratio of 3 WT versus 1 recombinant polyhedrin Figure A, and th(58–110)EGFP produced the brightest particles in our study, as assayed by flow cytometry, followed by fragments PH(1–110)EGFP and PH(1–58)EGFP, in that order. Most surprisingly, the less efficient was the full length polyhedrin (PH(1–245)-EGFP, Figure (58–110)EGFP as the most efficient of all. The reasons behind the increased incorporation of PH(58–110)EGFP remains unsolved but are a target of a current study. Most interestingly, this was the minimum fragment that could form self-aggregates, thus a hint about the greater efficiency of incorporation into polyhedra may reside here.Noteworthy, not all fragments that incorporate into polyhedra result in equal amounts of fluorescence emitted by the EGFP incorporated into the particles. Most notably the fragment PHrin  and PH(58–110) are electron dense particles, as identified in our TEM studies  as a powerful tool to produce high yields of recombinant protein that can be easily isolated by slow speed centrifugation from Sf9 cell lysates.The identification of the amino acids 58–110 as the smallest fragment capable of self-aggregation and efficient incorporation into polyhedra crystals from the N-terminal of AcMNPV is important in the development of new technologies to produce nanoparticles of interest in science and biotechnology, carrying foreign proteins of interest. Most interestingly, the self-aggregates produced by fragments PH Figures A and 6C, Figures B-C. Thes(58–110)) consisting of two beta domains followed by an alpha helix, which contains a self aggregating domain essential for polyhedra-like particle formation. This deletion strategy allowed also the identification of a nuclear retention signal in polyhedrin, contained within the first 110 amino acids. Even though the fragment PH(1–58) contains also two beta domains followed by an alpha helix, is not sufficient for self-aggregation, since the expression of this fragment results a soluble form contained in the cytosol of infected cells.Using a deletion strategy based on the crystallographic structure of AcMNPV polyhedrin protein, we have identified a minimum sequence (PHin vivo protein crystallization.These findings open new avenues to explore how polyhedra crystals are formed, and to understand what structural features may be required for


We report experimental measurements of electronic Raman scattering by electrons (holes) in individual single-walled carbon nanotubes (SWNTs) under resonant conditions. The Raman scattering at low frequency range reveals a single particle excitation feature. And the dispersion of electronic structure around the center of Brillouin zone of a semiconducting SWNT  is extracted. Many experimental techniques aiming at charactering electronic properties surrender in intrinsic one-dimensional systems particularly single-walled carbon nanotubes (SWNTs) owing to their atomic size and richness of geometric structures. Typical examples include the magneto-electric transport techniques and angle-resolved photoemission spectroscopy which informatively probe the band dispersion and electron (hole)'s quantum states in 3D and 2D materials, but lose ground in SWNTs. As yet, there lacks a method capable of directly evaluating the band dispersion in SWNTs. Resonance Raman spectroscopy has been recognized as one of the most powerful and popular characterizing techniques in SWNT research. SWNTs' geometric structures could be quantitatively identified at single nanotube level with the well-established protocols in resonance Raman spectroscopy13iiM excitonic transition energyRecently Farhat et al. reported a mode of electronic Raman scattering (ERS) from metallic SWNTs, where the Raman shift changes with the excitation energy and the scattered photon is exactly energetically resonant with the There exist several obstacles toward the observation of ERS in SWNTs: (i) The scattered lights are overwhelmed by Rayleigh scatterings from the substrate and SWNTs, and inelastic scatterings by the lattice vibrations at finite temperatures. The intensity of the ERS is several orders of magnitude weaker than that of Rayleigh scatterings; (ii) The ERS originating from single particle and collective excitations is usually around a few meV in terms of energy shifted from the excitation laser. It is technically challenging to distinguish such a low frequency Raman signal from Rayleigh scattering; (iii) Resonance Raman scatterings from individual SWNTs in most experimental setups are conducted with a confocal-like micro-Raman setup in order to maximize the light collecting power, where the incident light and scattered light are both at normal direction with respect to the SWNT axis. The momentum of the incident and scattered photons are both orthogonal to that of electrons (holes) and therefore the photon and electronic elementary excitations are decoupled at normal incidence due to 1D nature of SWNTs. Here we report our observation of electronic Raman scattering around 1 meV from a small suspended semiconducting SWNT bundle. We study the low frequency Raman spectra on SWNTs under the resonant excitation at oblique incident angles. The energy of the ERS shows linearly proportional to the momentum exchange with the interacting photons along the direction of the SWNT axis. We attribute the ERS to the single-particle excitation, and the energy-momentum dispersion of the resonant band could be directly probed.3 on the silicon substrate and then by being reduced under Ar/H2 400 SCCM/50 SCCM at 900°C for 20 min. Individual SWNTs were grown on the substrates in ethanol vapor with the same gas mixture at 900°C for 1 h. For the Raman scattering experiments, the incident light from a single transverse mode HeNe laser (633 nm) was focused on SWNTs through a 10× objective at oblique angles against the SWNT axis and the scattered light was collected by a 50× objective, as sketched in 3 W/cm2, and the acquisition time was 1800 s. The low frequency Raman signals was analyzed with a single stage monochromator  with a set of Brag-GrateTM notch filters optimized at anti-Stokes side which demonstrates much higher throughput than dual or triple stage monochromators, with high Rayleigh rejection rate at low frequency range720 ~ 30 μm wide 1 mm long slits on silicon substrates were fabricated with a standard microelectromechanical (MEMS) process, including low pressure chemical vapor deposition (LPCVD) silicon nitride etching mask growth, optical lithography, reactive ion etching (RIE) and wet etching. The SWNTs were in situ synthesized by chemical vapor deposition (CVD) across the slits. The catalyst was prepared by selectively dipping the diluted solution of FeClNe laser 33 nm was−1 with linewidth −1, as well as −1, 1581 cm−1 and 1585 cm−1 indicates that the sample is a small bundle of semiconducting SWNTs9d = 228/RBMω (nm ·cm−1) between nanotube diameters and the inverse of their Radial Breathing Mode (RBM) frequencies of suspended SWNTsRBMω = 125 cm−1) and 2.2 nm (RBMω = 106 cm−1). The interband transition iiS is determined by Rayleigh scattering spectroscopy. As shown in CCa = 0.142 nm is the nearest-neighbor C-C distance)33S of SWNT with chiral indices , while the one around 1.87 eV corresponds to the transition 44S of nanotube .Characteristic resonance Raman modes and Rayleigh scattering spectra are used to identify the sample's geometric structure, as demonstrated in ik cos α, the projection of the incident photon wave-vector along the SWNT axis, yielding the slope of 5.6 ~ 6.3 × 10−5 meV·cm. To our knowledge, the lowest frequency of phonon modes around Γ-point is in the range of 10 cm−1 ~ 12 cm−1−1. Besides, the slope of the acoustic phonon dispersion near Γ-point is estimated to be 4 ~ 16 × 10−7 meV·cm2–103 W/cm2, which modulates the effective carriers to some extent. Thus the bump unlikely originates from the Plasmon or other collective modes, as the corresponding dispersion is a monotonic function of effective carrier density but the observed features are independent of the excitation intensity. Besides, Rayleigh scattering implies that the contribution from SWNT  can be ignored as the excitation energy (1.96 eV) is away from the resonance conditions at low frequency Raman energy. So we attribute the low frequency mode to the single particle excitation (SPE) of SWNT , as illustrated in the inset of iiS relax and form excitons, a kind of quasiparticles in a time scale of picoseconds, while the ERS occurs almost instantaneously. So the ERS signal here reflects the properties of electrons (holes) instead of excitons. In the experimental setup, the incident angle concludes the photon's momentum projection ik cos α along the SWNT axis and therefore determines momentum transfer between the incident and scattered photons. As the requirements of energy conservation and momentum conservation along the SWNT axial direction, the energy difference between the incident and scattered photons exactly reflects the electronic band dispersion at resonance energy. Thanks to the perfect electron-hole symmetry for suspended SWNTs, the SPE simply probes the dispersion of the corresponding valence bands and conduction bands. This picture is qualitatively consistent with the fact that the Raman frequency increases with the increase of wave-vector transfer. As the magnitude of the wave-vector transfer is quite small (3 and V3) is calculated to be in the range of 5.6 ~ 6.3 × 10−7 meV·cm with the axial wave-vector Δk ranging from 9673 cm−1 to 13473 cm−1 in our experiments.Note that the hot electron-hole pairs generated in the optical transitions 33 transition. Given the excitation of 1.96 eV (633 nm) in our experiments and the empirical dispersion, we derive the corresponding wave-vector on the electronic energy band of SWNT  to be 1.446 × 107 cm−1, and the slope at this point to be about 1 × 10−4 meV·cm. The calculated result shows a qualitative agreement with what we observed in the experiments. On the other hand, if we follow the model of exciton Kataura plots with environment corrections1633 transition of SWNT  is now associated with the peak around 1.91 eV, so the slope −5 meV·cm. This is qualitatively consistent with our experimental results, which provides a strong support for our explanation of SPE energy dispersion picture.To qualitatively examine our experimental results, we apply the empirical formula−7 meV·cm on SWNT with structural index .In summary, we report a low-frequency Raman mode from semiconducting SWNTs under resonant excitations at oblique incidence. The corresponding Raman mode shows a linear dependence on the momentum transfer along the SWNT. We attribute the new Raman mode to the inelastic scattering arising from electronic single particle excitation (SPE). The slope of electronic band dispersion at the subband edges is measured around 5.6 ~ 6.3 × 10X.D.C. conceived and designed the experiments; X.C., B.Z. and A.Z. conducted the experiments; X.C., B.Z., H.Z., Q.Z. and X.D.C. analyzed and interpreted the data; All authors discussed the results and wrote the manuscript.


Integrating biological and computational modeling approaches can overcome this limitation. Here, we generated a biologically driven discrete hybrid cellular automaton (HCA) model of bone metastatic prostate cancer to identify the optimal therapeutic window for putative targeted therapies. As proof of principle, we focused on TGFβ because of its known pleiotropic cellular effects. HCA simulations predict an optimal effect for TGFβ inhibition in a pre-metastatic setting with quantitative outputs indicating a significant impact on prostate cancer cell viability, osteoclast formation and osteoblast differentiation. In silico predictions were validated in vivo with models of bone metastatic prostate cancer (PAIII and C4-2B). Analysis of human bone metastatic prostate cancer specimens reveals heterogeneous cancer cell use of TGFβ. Patient specific information was seeded into the HCA model to predict the effect of TGFβ inhibitor treatment on disease evolution. Collectively, we demonstrate how an integrated computational/biological approach can rapidly optimize the efficacy of potential targeted therapies on bone metastatic prostate cancer.The ability to rapidly assess the efficacy of therapeutic strategies for incurable bone metastatic prostate cancer is an urgent need. Pre-clinical  Metastatic castrate resistant prostate cancer (mCRPC) typically manifests in the skeleton and is currently incurable1478910111213151819in silico BMU is 1000 μm × 1500 μm and is composed of bone, mesenchymal stromal cells (MSCs), precursor and adult osteoblasts, and precursor and mature multinucleated osteoclasts. The sequence and timing of resorption and bone formation that emerges from the model recapitulates the extensive literature and the interactions of the cells were carefully modeled around bone derived factors including RANKL and TGFβPreviously, our group generated a HCA based computational model of the bone modeling unit (BMU) that recapitulates the homeostatic sequence of bone resorption and anabolismin vivo using two models of osteogenic bone metastatic prostate cancer. Moreover, using patient specific information from bone metastatic specimens, we demonstrate the flexibility of the HCA model in predicting the efficacy of TGFβ inhibitors on lesions that are heterogeneous for TGFβ utilization. Collectively, we demonstrate how an integrated computational/biological modeling approach can be used to optimize therapy efficacy for the treatment of bone metastatic prostate cancer.TGFβ inhibitors such as neutralizing antibodies are currently undergoing clinical trial52324In silico, the ability of stromal cells to respond to varying TGFβ concentrations (0.1 to 10 ng/ml) was integrated into our HCA of normal bone remodeling, the bone multicellular unit (BMU)  at a dose previously used in the literature (10 mg/kg) and consistent with clinical trials performed for a humanized version of the 1D11 TGFβ neutralizing antibody, fresolimumab/GC10082229in vivo information, we applied the TGFβ inhibitor to the normal BMU at a level of 80% in silico. The stochastic nature of the BMU allows for variation and statistical analysis of simulation outputs. The results of multiple simulations (n = 29/group) show that TGFβ inhibition significantly promoted bone formation (9% increase) over a 75-day period by enhancing osteoblast expansion and differentiation while limiting osteoclast viability  see 18. To obiability . ImportaThe vicious cycle paradigm suggests that metastatic prostate cancer cells utilize TGFβ signaling to promote their survival and growth. We therefore seeded the computational model with TGFβ responsive prostate cancer cells. Once the vicious cycle was established at day 80, we initiated TGFβ inhibition (post-treatment scenario). Simulations (n = 24/group) revealed that TGFβ inhibition reduced cancer growth by approximately 15%, but only when the inhibitor was applied at a constant 99% level of efficacy until day 250 . At a moin silico at day 1 prior to the seeding of the cancer cells (pre-treatment scenario) even at a level of 20% efficacy significantly reduced tumor burden over time by ≥65%  revealed that applying the TGFβ inhibitor  by ≥65% . Interes by ≥65% , Day 250In vitro analysis of prostate cancer cell lines that can grow in the bone microenvironment identified the PAIII cell line as reflecting the TGFβ receptor and growth factor producing (TRP) status observed in human specimens  show that TGFβ ligand and receptors are expressed and pSMAD2 staining indicates that the TGFβ pathway is active . In vitrpecimens . We alsopecimens . We therin silico illustrates TGFβ inhibition directly limits growth over time by impacting cancer cell proliferation  with a TGFβ inhibitor would not be of benefit. Our in vivo data confirm these predictions  less tumor induced osteolysis in the TGFβ inhibitor group compared to the controls as determined by X-ray , the computational model predicts TGFβ inhibition would ultimately result in decreased bone formation , we also noted the presence of cancer cells that either produced the ligand alone (TP), the receptor alone (TR) or neither (TN) . This rae groups .In silico data show the effects of pre- and post- TGFβ inhibitor treatment on clonal evolution for this patient over time and the responses of the surrounding tumor microenvironment  samples are being used to design precision treatment strategies and integrating the biological parameters derived from these specimens into computational models could prove to be a synergistic way to tackle the complexity of heterogeneity in individual patientsIn conclusion, we have developed a novel and unbiased computational HCA model that allows for the dynamic multi-scale understanding of how metastatic prostate cancer cells evolve and interact with the surrounding bone microenvironment. We used this model to predict the efficacy and response of bone metastatic prostate cancer to targeted therapies such as, TGFβ inhibitors. Further, our integrated computational and biological approach allowed for the dissection of how TGFβ inhibition simultaneously affects osteoblast, osteoclast and cancer cell behavior over time. The HCA model constitutes a platform for discovery that can readily be expanded to incorporate additional cellular and molecular circuitry. This will ultimately yield a clinical tool that will aid the medical oncologist in designing curative strategies for heterogeneous bone metastatic prostate cancer. Most importantly, this modeling approach can be applied to the development of new therapeutic strategies across a broad spectrum of human malignancies.Log(TGFβ)) or inversely proportional (−1Log(TGFβ)) manner. TGFβ inhibition in the HCA is achieved by controlling TGFβ bioavailability (0–99% inhibition at a constant level). Computational models were seeded with homogeneous or heterogeneous prostate cancer cells that expressed the TGFβ receptor and ligand (TRP), the ligand alone (TP), the receptor alone (TR) or were negative for both (TN). For the HCA model, we consider cellular intrinsic behaviors and the impact of TGFβ on these behaviors. We include 8 different cell types: resident and active cells of the bone stroma , precursor osteoblasts (pOB), adult osteoblasts (aOB), precursor osteoclasts (pOC), adult osteoclasts (aOC)) and prostate cancer cells of varying TGFβ responsiveness . We considered interactions between all cell types and the impact of these interactions on the tumor-bone microenvironment. Empirical, experimental and theoretical parameters were used to fuel equations . If the levels of TGFβ are below the saturation level, the probability of death for the aOC increases56where 0 < TGFβ < 1 and BDN) for their division. The probability of division was estimated as being proportional to the inverse logarithm of the available BDN. If there are no nutrients, there is zero division. If there is maximum nutrient saturation, the division rate is at its maximum.Prostate cancer cells were explicitly defined as being dependent on TGFβ and bone derived nutrients  soft agar assays . TRP divDivTRP is the maximum rate of TRP division, in the saturation level of bone derived nutrients. Under TGFβ inhibition, the maximum division rate is assumed to be reduced for TRP to once every 2 days based on empirical observations in our laboratory (data not shown).where 0 < TGFβ < 1 and We assume that TR has a maximum division rate of 1.75 days, but a lifespan without nutrients of 10 days, calculated as not having a cost for producing TGFβ ligand but benefiting from the presence TGFβ in the bone microenvironment. TR cell division depends directly on bone-derived nutrients in the same manner:DivTR is the maximum rate of TR division, in the saturation level of bone derived nutrients. Under TGFβ inhibition, the maximum division rate is reduced for TR to once every two days.where 0 < BDN < 1 and We assume that TP has a maximum division rate of once 1.75 days, and a lifespan without nutrients of 10 days, calculated as having a cost for producing TGFβ ligand but not benefiting from TGFβ contained within the bone derived nutrients. TP cell division depends directly on bone-derived nutrients in the same manner:DivTP is the maximum rate of TP division. Under TGFβ inhibition, the maximum division rate of TP remains the same.where 0 < BDN < 1 and Finally, we assume TN has a maximum division rate of once every 2 days and a lifespan without nutrients of 12 days, calculated from having no cost of producing TGFβ and no benefit from TGFβ signaling. TN cell division depends directly on bone-derived nutrients through:DivTN is the maximum rate of TN division. Under TGFβ inhibition, the maximum division rate of TN remains the same.where 0 < BDN < 1 and Luciferase-expressing PAIII, C42B, and PC3 prostate cancer cell lines were cultured in complete Dulbecco’s Modified medium supplemented with 10% fetal bovine serum5859Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Pre-Treatment Studies: 6-week old male SCID Beige mice were injected intraperitoneally with either TGFβ inhibitor  or isotype control, 13C4 , a kind gift from Scott Lonning and Patrick Finn at Genzyme. Subsequently, luciferase expressing PAIII or C4-2B cell lines  were intratibially injected either one-day or one week after TGFβ pre-treatment61All animal experiments were performed with IACUC approval (R1283) and were conducted in accordance with the guidelines set forth in the A detailed description of cell assays, histological and bone morphology analyses can be found in How to cite this article: Cook, L. M. et al. Predictive computational modeling to define effective treatment strategies for bone metastatic prostate cancer. Sci. Rep. 6, 29384; doi: 10.1038/srep29384 (2016).


It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, β2GPI and DI in APS.Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays.Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3–5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS.All assays displayed good specificity for APS; IgG aCL and IgG aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.Measuring IgG aDI and IgA aβ The ELISAs measure IgG and IgM aPL, while the LA test does not discriminate between antibody isotypes  versus subjects with aDI [aCL/aβ2GPI+(aDI+)] ] .2GPI, 52 of which were also IgG aDI-positive; 52 subjects had IgM aCL/aβ2GPI of which 33 were IgM aDI-positive; and 100 subjects had IgA aCL/aβ2GPI of which 38 were IgA aDI-positive. For all isotypes, the presence of aDI increased the HR for APS by approximately 3-fold for IgG and IgM, and 5-fold for IgA  and aCL/aβ2GPI+(aDI+) serological profiles (2GPI+(aDI-)  and IgG aCL/aβ2GPI+(aDI+)  were associated with VT. No significant associations were seen for IgM or IgA serological profiles, except that the addition of IgA aDI positivity tripled the HR for thrombosis and converted it from non-significant (HR for thrombosis in aCL/aβ2GPI+(aDI-) subjects 1.1, 95%CI 0.4–2.9) to significant (HR for thrombosis in aCL/aβ2GPI+(aDI+) subjects 3.6, 95%CI 1.4–9.1). PM was not associated with either the aCL/aβ2GPI+(aDI-) or aCL/aβ2GPI+(aDI+) profile . Of interest, LA positivity alone could not discriminate between thrombotic or obstetric complications in our APS cohort .The same approach was applied to the group of 111 patients with APS in order to establish the HR for thrombosis or pregnancy morbidity associated with the aCL/aβprofiles . Both Ig profile . These f2GPI and aDI in patients with APS, SLE and healthy controls. We confirm the importance of IgG aCL and IgG aβ2GPI tests, which had the highest sensitivity for APS and were strongly associated with LA positivity. We show that IgA aβ2GPI are strongly associated with APS and are more common in our cohort than IgM aβ2GPI, and demonstrate that aDI of all three isotypes are associated with APS with high specificity. Importantly, in subjects known to be positive for any isotype of aCL and/or aβ2GPI, the additional finding of aDI positivity increases the likelihood of APS by between three and five times. Finally, we report that positivity for IgG or IgA aDI increases the strength of association between aCL/aβ2GPI and thrombotic manifestations in APS.In this study, we performed nine different assays using sera from 430 subjects. This large dataset allows the first rigorous comparison of IgG, IgM and IgA aCL, aβ2GPI is well characterised both in vivo and in vitro [2GPI have been far less studied in comparison [2GPI, highlighting the low quality of the data, variability of results and that many studies had been restricted to patients with SLE [2GPI) in patients who are IgG/IgM- and LA-negative but in whom APS is suspected [2GPI and aDI would be more valuable than measuring IgA aCL in patients with suspected APS, and we also note that IgA aβ2GPI are more commonly positive in our APS cohort compared to IgM aβ2GPI . We tested a total of 32 serum samples– 13 APS; 8 SLE/no APS; and 11 healthy controls—in both our in-house and the Inova assays, and found that the two assays showed very good quantitative  and qualitative agreements .While the pathogenicity of IgG aβin vitro –9, IgA amparison , 5. In awith SLE . Due to with SLE , yet somuspected . Our datM aβ2GPI . To conf2GPI to recognize aPL when immobilised on a plastic surface relies on its conformation and is a significant obstacle towards standardising aβ2GPI tests across laboratories. In contrast, we and others have successfully used recombinant human DI expressed in bacteria [2GPI. A number of different assays have been reported for measuring aDI , demonstrating that both aDI and aDIV-V can be detected in the blood of aβ2GPI-positive subjects. Importantly however, aDI were more frequently found in patients with systemic autoimmune disease compared to asymptomatic aPL carriers [The ability of human plasma purified βbacteria , baculovbacteria , and synbacteria  to measubacteria , 43–45, iewed in ). Thus fiewed in , 21, whiiewed in . In a smy of 46% . Likewisd to aDI . MoreoveDI  but high IgG aDI (>50GDIU). One of these patients also had high IgM aDI (>100MDIU) despite low IgM aCL (20MPLU) and IgM aβ2GPI (21MBU). Therefore, although rare, there are patients with APS with high aDI activity but low or negative classical IgG/IgM aPL activity, and in these cases aDI tests could certainly provide additional information to current tests available. Of relevance, we recently reported the presence of IgG aDI in 3 out of 40 ‘seronegative’ APS patients, who fulfilled clinical but not serological APS criteria [In line with recently published studies , 45, we positive . Interescriteria , further2GPI-positive subjects (of which 82% had APS), underlining the added value of measuring IgG aDI as well as aCL/aβ2GPI in relation to increased risk of VT [et al [2GPI-positive subjects [Our results concur with the largest published study for IgG aDI in 442 aβsk of VT . We addiT [et al  however,subjects .2GPI, since the latter would also pick up aβ2GPI directed against other domains. Considering their pathogenic role, detecting IgG aDI may allow for risk stratification in established APS and help in the diagnosis of suspected APS, while the prevalence and clinical association of IgA aDI in APS requires further clarification. Moreover, prospective studies are imperative to determine if IgA aβ2GPI and IgG/IgA aDI have prognostic power. We are currently completing such a study in early samples from >500 patients with SLE, a proportion of whom later developed thrombosis or obstetric complications, and are performing longitudinal tests in order to establish if aPL levels remain constant or change before, near to and after an APS-related clinical event.Based on our current findings and other groups’ published reports, we consider aDI tests as a useful additional test rather than a replacement for tests using whole β


In fluorescence microscopy, the distribution of the emitting molecule number in space is usually obtained by dividing the measured fluorescence by that of a single emitter. However, the brightness of individual emitters may vary strongly in the sample or be inaccessible. Moreover, with increasing (super-) resolution, fewer molecules are found per pixel, making this approach unreliable. Here we map the distribution of molecules by exploiting the fact that a single molecule emits only a single photon at a time. Thus, by analysing the simultaneous arrival of multiple photons during confocal imaging, we can establish the number and local brightness of typically up to 20 molecules per confocal (diffraction sized) recording volume. Subsequent recording by stimulated emission depletion microscopy provides the distribution of the number of molecules with subdiffraction resolution. The method is applied to mapping the three-dimensional nanoscale organization of internalized transferrin receptors on human HEK293 cells. Mapping the distribution of fluorescence molecules, rather than just their emission intensity, can improve super-resolution fluorescence microscopy. Here, the authors present a general solution for rendering the number of fluorescent molecules recorded by confocal or STED microscopy. Ideally, a microscope discerns and maps all features and molecules of interest in the sample. Throughout the twentieth century it was widely accepted that any attempt to develop such a microscope would require an electron or scanning probe type of approach, because discerning features that are closer together than about half the wavelength of light would not be possible using optical lensesOf major relevance to mapping the molecules is that this transient state difference can be elicited either in a spatially controlled (coordinate targeted) or in a spatially stochastic manner. The first strategy is realized in the methods called stimulated emission depletion (STED)36Spatially stochastic methods such as those called photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM) prompt adjacent molecules individually to a specific transient on-state in which the molecule is able to emit multiple fluorescence photons8910In contrast, STED microscopy works with standard fluorophores that do not require photoswitching between metastable states, because the on–off-based super-resolving ability is accomplished with the fluorophore's basic ground and fluorescent state. Being not based on single-molecule detection and by registering all molecules from a given coordinate simultaneously, STED microscopy provides a potential speed advantage, but the very same fact makes molecule counting more challenging. In principle, one can extract the number of registered molecules from the magnitude of the fluorescence signal, but in practice this approach is compromised by variations in molecular brightness due to the local environmental heterogeneity. In addition, in most cases, the local brightness of individual molecules remains elusive, because the sample does not contain isolated molecules, or such molecules are simply not found. A reliable method to reveal the number of molecules in coordinate-targeted nanoscopy is very desirable but still missing. Fortunately, the number of molecules is also encoded in the photon emission statistics, specifically in the number of photons that are emitted simultaneously. Measuring the simultaneous arrival of fluorescence photons has been shown to identify individual fluorophores13151718Here we explore the same effect of simultaneous photon arrivals in imaging mode and establish a theoretical model followed by a standard reconstruction procedure to retrieve the number of molecules with diffraction unlimited resolution.n(r) describing the number of molecules at coordinate r±Δr/2, with Δr denoting the spatial interval in which molecules can assume the on-state. Δr is equivalent to the spatial resolution given by the full-width at half-maximum of the effective point spread function (PSF) h(r) of the imaging process. Excitation is accomplished with illumination pulses much shorter than the fluorescence lifetime so that each molecule is excited at most once per pulse (see r are assumed to have the same brightness p(r), which is the probability of a molecule at the focal centre to yield a detected photon. p(r) takes into account the excitation intensity, the absorption cross-section, the fluorescence quantum yield and the collection efficiency of the setup. The ideal case p(r)=1 is reached if the molecule contributes with a detected photon for every pulse. If p(r) is temporally constant, the probabilities fi(r), i=1, 2, of recording i photon detection events per illumination pulse at coordinate r are:Mapping the molecule distribution can be reduced to finding a function ulse see . Moleculβ considers the detection geometry (see f2 is given by the probability of detecting two photons from any molecule minus the ‘antibunching term' (p2n)*h2, which accounts for the probability that the two photons stem from the same molecule. f2 depends quadratically on p(r), making it a critical parameter. If (np*h)(r)<<1 does not hold, for example, when the number of simultaneously recorded molecules is too large, etry see  and ‘*' f1 and f2 by detecting the number of one- and two-photon events divided by the number of pulses applied. The desired n(r) is ultimately extracted by fitting the measured f1 and f2 with the model from equation p(r) (The strategy is now to estimate ion p(r) .nest and the brightness pest of molecules in a single cluster can be derived analytically as M is the number of excitation pulses. Note that for increasing n, RSTD(nest) does essentially not depend on n. The calculations and simulations suggest that the RSTD of the estimations on both n and p can be below 20% under conditions provided by routinely used synthetic fluorophores, such as ATTO 647N and Abberior STAR 635P  of the estimated number ally as  , which wally as  . M is thTAR 635P . TherefoTAR 635P  so that To validate our prediction, we utilized double-stranded DNA (dsDNA) that is easy to synthesize and label in a controlled manner. Our first sample consisted of dsDNA labelled with up to four ATTO 647N or two Abberior STAR 635P fluorophores, sparsely immobilized on a glass surface. A pixel dwell time of 300 μs  and 20-nm-sized pixels of the confocal recording were adequate to collect 6,000–7,500 photons per fluorophore and a reduction of ∼30 per fluorophore in two-photon detection events because two-photon emission cannot come from the same molecule . Thus, aDistributing the dsDNA sparsely on the surface is not required for our method, but it facilitated counting the number of fluorophores on the dsDNA by bleaching them down individually. The number of individual bleaching steps matched the established number of fluorophores with a RSTD of <20% , which lOn STED illumination, the apparent size of the dsDNA becomes smaller as the resolution of the system improves. At the same time, the number of photon detection events per molecule decreases and the uncertainty (RSTD) of the molecule counting increases . To keepfi,c/s(r) means i  photon detection probability of confocal/STED microscopy, with hc/s denoting the corresponding effective PSF. To recover n and p, the fitting is now conducted by minimizing the distance from the model in i photon detection events  simultaneously in both confocal and STED measurements  calls for relatively high excitation intensities, but since high intensities increase blinking and bleaching, we settled for ∼0.1 pJ per excitation pulse giving ∼60% of the maximal emission  only if the number of simultaneously recorded molecules becomes lower, for example, <5. Our study also suggests that collecting the emitted fluorescence from both sides of the sample will facilitate the molecule counting in a pure STED mode at better resolution. Note that the effective PSF describing the imaging by simultaneous m-photon detectionmth-order antibunchingOur study reveals that counting the molecules in a confocal recording and allocating them using STED is a favourable strategy, because the counting accuracy benefits from increased resolution  with 100-ps pulse width for fluorescence excitation, and a mode-locked titanium–sapphire laser operating at 770 nm for STED . The repetition rate of the STED laser is 76 MHz and the initial 150-fs pulse width is stretched to about 200 ps using an optical fibre. The excitation and STED laser pulses are synchronized, but the excitation is triggered only at every 4th or 7th pulse to reduce the repetition rate to ∼20 or 10 MHz, respectively. The focal STED doughnut was created by passing the STED laser beam through a polymer vortex phase plate  and feeding it into a 1.4 numerical aperture (NA) objective lens . The fluorescence was collected by the same objective lens and filtered out by passive dichroic mirrors and filters . The fluorescence beam was split into four channels by three 50:50 beam splitters, coupled into four multimode fibres and detected by four avalanche photodiodes . The fibres acted as confocal pinholes and were aligned to the focal excitation maximum.The scanning, data acquisition and analysis software  were cod−1 biotinylated BSA for 30 min. After another washing step with PBS buffer, a solution of 0.5 mg ml−1 streptavidin  was used to incubate the coverslip for 30 min. Finally, the coverslip was washed again with PBS and was used for immobilization of the dsDNA (100 pM). ROXS buffer was used to increase the photostabilityThe dsDNAs with multiple ATTO 647N, Cy5 or Abberior STAR 635P molecules are formed by annealing from single-stranded DNAs (ssDNA), which were purchased directly from IBA , by heating the mixture of complementary ssDNAs to 95 °C and cooling it down to room temperature gradually in 45 min. The sequences of ssDNAs with four ATTO 647N molecules: biotin 5′-CGCCTATCCATAAACGAGGAGGACGCCTATCCATAAACGAGGAGGACGCCT ATCCATAAACGAGGAGGACGCCTATCCATAAACGAGGAGGA-3′ and 5′-TCCTCC TCGTT(ATTO 647N)ATGGATAGGCG-3′; the sequences of the ssDNAs with five Cy5 molecules: biotin 5′-ACACGACTTCTCCTCTCTCTCACGTTCCTTACCTACACCATCTTTACC TACTTACCCCACATCACTATCCCTTCTCATTCCTTTA-3′ and Cy5 5′-TAAAGG AATGAGAAGGGATAGT(Cy5)GATGTGGGGTAAGTAGGTAAA(Cy5)GATGGTGTAGG TAAGGAACGT(Cy5)GAGAGAGAGGAGAAGTCGTGT-3′; and the sequences of the ssDNAs with two Abberior STAR 635P: (biotin) 5′-TTATTCCTGTAGTATATGGCAATGAAA TTAT-3′ and 5′-ATAATTTCATTGCCATATACTACAGGAATAA-3′. The immobilization is performed by streptavidin–biotin linkage to the biotinylated bovine serum albumin -coated glass surface. In brief, the coverslip was cleaned with 2% Hellmanex II  and washed with PBS, pH 7.4, and, then, incubated with 1 mg mlThe DNA origami sheet was assembled from a long single-stranded scaffold strand from M13mp18 phage and ∼200 staple strands−1) DMEM supplemented with 10% fetal calf serum, 4 mM L-glutamine and 100 U ml−1 of each penicillin and streptomycin. For imaging, cells were grown on poly-L-lysine-coated glass coverslips and typically stained 16 h after seeding.HEK293 cells were cultured in high glucose  in PBS followed by 10-min incubation with 250 μM nuclear staining dye SYTOX green . Finally, cells were rinsed in PBS and mounted in Mowiöl mounting media . Confocal images were obtained using a TCS STED SP5 fluorescence microscope (Leica Microsystems GmbH), equipped with a 1.4 NA/63 × STED objective (Leica). Confocal sections with pinhole diameter set to 1 Airy disc were acquired to cover the complete volume of individual cells. Total cellular volumes and nuclear volumes were calculated by manual selection of cellular or nuclei contour (in focus) for every optical section (using ImageJ). The calculated areas of each slide were multiplied by the section thickness (0.797 μm) and summed up to obtain the volumes of the whole cell and nucleus. Cytosolic volume was obtained by subtracting the nuclear volume from the total cell volume. Average cell volume was obtained from 10 individual cells.HEK293 cells seeded on glass coverslips were chemically fixed in PBS supplemented with 4% paraformaldehyde (PFA) for 30 min at room temperature. Quenching of the remaining reactive formaldehyde groups was performed by incubation for 15 min in PBS supplemented with 0.1 M glycine and 0.1 M NHL-lysine-coated coverslips were fixed in 4% PFA for 45 min at room temperature, washed with PBS. Remaining formaldehyde was quenched with 0.1 M glycine and NH4Cl in PBS for 15 min. To perform a complete immunostaining that also labels the internal epitopes, coverslips were incubated with 2% BSA and 0.1% Triton X-100 in PBS for 15 min at room temperature. To detect only the TfRs present at the cell surface, cells were incubated in 2% BSA in PBS (without detergent). Primary antibody anti-TfR  was used at a final concentration of 10 μg ml−1 in PBS with 2% BSA for 60 min at room temperature. This monoclonal mouse antibody was chosen since it targets an extracellular epitope of the TfR, allowing us to perform the staining of surface molecules. After thorough washing steps, primary antibody was detected by Cy3-coupled anti-mouse secondary . Finally, coverslips were rinsed in PBS and mounted in Mowiöl (see above). Epifluorescence images were acquired with an Olympus IX71 microscope equipped with 0.75 NA/40 × objective and an Olympus F-View II CCD camera . Image intensities of individual cells were measured by manual selection of cellular contour (ImageJ). Average intensities for surface and complete immunostaining were obtained from three independent experiments with 30–60 cells analysed per experiment.HEK293 cells on poly-2, heated up in a thermocycler to 75 °C for 3 min, and then cooled to 20 °C at a rate of 1 °C min−1 to achieve the active conformation/folding of the aptamer. HEK293 cells were washed briefly with pre-warmed Ringer buffer  and incubated in cell medium (see above) for 30 min at 37 °C with 1 μM of ∼5 kDa dextran sulfate and 1 mg ml−1 of sheared salmon sperm DNA (Invitrogen) as pre-blocking agents. Cells were incubated in 60 μl of aptamer c2 at a concentration of 0.1 pmol μl−1 (∼3.6 × 1012 molecules) for 60 min (saturating staining) at 37 °C in complete medium supplemented with 1 mg ml−1 of sheared salmon sperm DNA. Cells were finally washed extensively with ice-cold PBS and fixed with 4% PFA as above. Following quenching of the fixative, cells were briefly rinsed and left in ROXS bufferAptamer stainings were performed as described earlier7 cells per ml in RIPA Buffer (Sigma-Aldrich) that was complemented with 1 mM EDTA and protease inhibitors  using a cell scraper. For further homogenization, the lysates were vortexed, sonicated and passed through a 1-ml syringe fitted with a 23-gauge needle. After quantification of the protein concentration, cell lysates were snap-frozen in liquid nitrogen and stored at −80 °C. For protein separation, cell lysate samples were mixed with SDS sample buffer  and subject to gel electrophoresis using 10% denaturing Tris/Tricin SDS polyacrylamide gels in a discontinuous buffer system . Following the gel electrophoresis, western blotting onto nitrocellulose membrane and immunolabeling were performed as described beforein vitro expressed recombinant full-length human TfR  was used as a standard. To create a standard curve, the recombinant protein was prepared in concentrations increasing from 0 to 50 ng. Fetal calf serum was added to the recombinant protein in amounts matching the total protein concentration in the HEK293 cell lysate to have equal total protein concentration in all lanes. Linear regression was applied to the linear portion of the standard curve to determine the absolute amount of the TfRs in the cell lysate. Apparent molecular weight of the protein (98 kDa) including the post-translational modifications was preferred in the calculations rather than the theoretical molecular weight of the peptide sequence.HEK293 cells were collected and lysed at a concentration of 2 × 10How to cite this article: Ta, H. et al. Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6:7977 doi: 10.1038/ncomms8977 (2015).Supplementary Figures 1-15, Supplementary Note 1 and Supplementary ReferencesA 3D molecular map of the TfR (stained with aptamer in HEK293 cells) is reconstructed from the photon statistics of confocal and STED measurements. Isosurfaces of the molecular map which embrace 70% of the overall number of molecules in the corresponding cluster are plotted in the animation. The cluster segmentation is performed with the built in watershed function in MATLAB. 0 - 5 s: reconstruction from the photon statistics of confocal recording (Red). 6 - 11 s: reconstruction from the photon statistics of confocal and STED recording (Cyan). 19 - 31 s: reconstruction from the photon statistics of confocal and STED recording with color code representing the number of molecules in each cluster. The full region is 8 x 8 x 2 μm.This software package contains the algorithm for molecular counting based on photon statistics in Confocal and STED microscopy.


In this paper, we consider the existence of least energy sign-changing solutions for a class of Kirchhoff-type problem  We denote by Next, we give some notations. Let mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimg satisfying asymptotically 3-linear condition. Very recently, combining constraint variational methods and quantitative deformation lemma, Shuai  is a critical point of We assume that mentclass2pt{minimNext, we further give the following properties of For any fixedwithhas a unique critical pointwithwhich is the unique maximum point ofonFor any Step 1 The existence of critical points for (i)(ii)t large and s large.Here we only prove n large, it has Firstly, for any fixed hand, by  and 6),,\documenation of  and 7)  \documenmentclasspt{minimamentclasspt{minimamentclasspt{minimaNext we show that the property (ii) holds for Applying  again, iT is continuous on By the property (ii), there exists Step 2 The uniqueness of critical point for From the Step 1, Case 1: Case 2: Step 3in Shuai , so we ox in Shuai (g.Throughout of the proof, making use of some subtle analytical skills instead of Implicit Function Theorem used in Shuai , we onlyin Shuai . Hence, From Lemma Ifthenthat is,Now we can prove Theorem  mentclass2pt{minimUsing Lemma In order to establish the property of the energy of any sign-changing solutions is larger than twice that of the ground state solutions of , we alsoFor any fixedthere exists a uniquesuch thatWe consider the functionx,λ≥0.By , we concX due to Corollary 2.9 in He and Zou (Note that  and Zou . Similar and Zou , we can mentclass2pt{minimFor mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimOn the one hand, using some subtle analytical skills and relaxing in Shuai  to \documentclass2pt{minimmentclass2pt{minimtions of , respect


The immunological response during the first 24 hours after traumatic brain injury (TBI) may be a critical therapeutic interval for limiting the secondary neuronal damage that is influenced by enhanced inflammatory mediator expression.ex vivo mouse cortical slice injury system and an in vivo piglet model of brain injury.To gain further insight of the early injury response, we examined the expression of several inflammatory genes by real-time qPCR as a function of time or distance from injury in two distinct mammalian models: an Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), chemokine ligands 2 (CCL2), 3 (CCL3), 4 (CCL4), and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNAs increased within 5 h after injury in mouse cortical slices. Chemokine and PTGS2 mRNAs remained elevated in slices at 24 h, whereas IL-1β and TNF-α expressions decreased from earlier peak levels. At 24 h after cortical injury in 1-month-old piglets, the expression of CCL2 mRNA was significantly increased in the lesion core and in the penumbra region. The expression of PTGS2, IL-1β, and TNF-α was variable among the piglets.in vitro and large animal models of cortical injury expand our understanding of the early timing and spread of the immunological response and can serve as preclinical systems to facilitate the discovery of therapeutic agents for TBI aimed at regulating inflammatory mediator expression.These  Traumatic brain injury (TBI) is a major health concern in contact sports and in the military and is the leading cause of death and disability in children and young adults. Though progress has been made in prevention and acute clinical management, a therapeutic that ameliorates the disability or mortality associated with TBI remains elusive. Despite the growing awareness of this public health problem, a greater translational effort in clinical trials for TBI is needed . ExpandiSecondary damage in surrounding brain tissue occurs following the primary insult. Pro-inflammatory mediator expression is enhanced soon after injury and is followed several days later by a period of enhanced expression of anti-inflammatory cytokines . While tin vivo rodent models of TBI. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increase within the first several hours after cortical injury but then return to baseline levels by 24 h [The time course of inflammatory mediator expression is well-established in  by 24 h . Chemoki by 24 h -9. IL-1β by 24 h ,8,10.While most of our understanding of the early expression and effects of inflammatory mediators in response to cortical injury is from the rodent models of TBI, the timing and contribution of long-term deficits in larger animal models and in human patients are less clear. An increase in TNF-α and IL-1β is detected in human cortical tissues early after injury . An increx vivo changes in the mRNA expression of cytokines, chemokines, and PTGS2 that occur during the first 24 h after injury. Slices maintain neural cells in their normal microenvironment, and an acute innate immune response occurs following tissue sectioning. In the second system, we used piglets to assess the in vivo changes in the expression of these inflammatory genes 24 h after cortical impact at the lesion core and in the penumbra region. The piglet brain is gyrencephalic where the abundance of white matter, anatomy, vasculature, and pattern of development closely parallels humans [Discovering new therapeutic agents with ideal immunomodulatory characteristics in the CNS may improve outcomes when administered early after TBI. This discovery effort will be facilitated by filling the gaps in preclinical translational research that exist between cell culture and rodent models and between rodent models and human studies. To fill these two gaps, we examine the early changes in the mRNA expression of inflammatory mediators following cortical injury in two understudied systems of TBI. In the first system, we use mouse cortical slices to evaluate the s humans . These tn = 8) were obtained from Jackson Laboratory  and housed for several weeks at the Borwell facility in Dartmouth’s Center for Comparative Medicine and Research. Acute cultures of cortical slices were prepared as described previously in more detail [TM solution.The Institutional Animal Care and Use Committee at Dartmouth College approved all mouse protocols. Adult (2 to 3 months old) female SJL/J mice (e detail  with mine detail . Slices n = 6) were obtained from Earle Parsons & Sons, Inc.  and were housed in the MGH Center for Comparative Medicine. Cortical impact has been described previously in more detail [2, and oxygen saturation were kept within a narrow range for the duration of the surgery. A craniectomy was performed at the junction of the right coronal and sagittal sutures, and the dura incised to expose the cortical surface. Cortical impact to the rostral gyrus was achieved via a spring-loaded device secured to the skull, and an indentor tip scaled to 1% of the volume of the brain was displaced over 4 msec. In injured animals, this produces a lesion size of 350 ± 50 mm3 or approximately 10% of the volume of the contralateral hemisphere 1 week after injury that typically extends down into the gyral white matter [3) were obtained immediately from the lesion core, penumbra, 2 cm anterior to the injury, and corresponding cortical region from the contralateral hemisphere. The lesion core was the region directly under the indentor tip. The penumbra was the region surrounding the lesion core. Samples were divided and/or snap frozen on dry ice, stored at −80°C freezer, and then immersed in RNAlaterTM solution prior to RNA isolation.All protocols were in accordance with the guidelines of the American Veterinary Association and the National Institutes of Health (NIH) and were approved by the Animal Care and Use Committee of Massachusetts General Hospital. One-month-old male Yorkshire piglets  method using β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.12) as internal control reference mRNAs [n = 6) was analyzed in triplicate and expressed as mean fold difference ± SEM relative to baseline levels (time 0) in a freshly isolated cortex. In the piglet cortical injury experiment (n = 6), cDNA from individual lesion core, penumbra, 2 cm anterior, and contralateral regions were analyzed in triplicate and expressed as average fold difference ± SEM relative to average levels in contralateral tissues. For analysis of mRNA changes in individual piglets (n = 6), CCL2 and PTGS2 (EC 1.14.99.1) mRNAs in penumbra and lesion core regions were expressed as fold difference relative to levels in the contralateral tissue from the same piglet.After removing excess RNAlater™ solution from the piglet or mouse cortical tissues, RNA was extracted using TRIzol Reagent . The eluted RNA was quantified by spectrophotometry, and 2 μg was reverse-transcribed using qScript cDNA SuperMix . Mouse oligonucleotide primer sets were described previously . Piglet ce mRNAs . In the t test was used for comparisons of ipsilateral cortical regions relative to uninjured contralateral regions in the piglets. The correlation between the mRNA fold changes among individual piglets was analyzed by a linear regression model. Statistical significance was defined as P < 0.05.GraphPad Prism Software  was used to generate graphs and conduct statistical analysis. One-way ANOVA followed by Dunnett’s multiple comparison test was used for comparisons of various incubation times relative to baseline amounts in mouse slices. Student’s To analyze the transcriptional changes in cortical slices, abundance of mRNA was measured in slices incubated for 0.5, 4.5, 6, and 24 h. Freshly isolated cerebral cortices were used as the 0 h baseline values. IL-1β and TNF-α transcripts increased from 0 to 6 h and then decreased between 6 and 24 h (Figure We analyzed next the level of inflammatory transcripts in piglet cerebral cortices after a cortical impact. Tissues from lesion core, penumbra, and 2-cm anterior to the penumbra were collected 24 h after injury for comparison to cortical tissue collected from the corresponding region on the uninjured contralateral side. While GAPDH and β-actin are both stable endogenous reference genes in uninjured pig cerebral cortex , we founSome genes are found to be less stable across individual outbred pigs even in uninjured cerebral cortex . Therefoin vivo rodent models. Alternatively, glia are activated in vitro with endotoxins or cytokines in dissociated cell culture systems. Yet, therapies identified in current model systems have not easily translated to clinically effective treatments [in vivo rodent models.The immunological response to neural injury is predominantly investigated in the eatments . Additioex vivo has the advantage of maintaining glia in their neural microenvironment, and the sectioning of tissue causes an immunological response without the requirement of endotoxins. While the change in acute expression of inflammatory mediators in spinal cord slices reveals a similar profile to observations in spinal cord injury in the rodents in vivo [in vivo models of TBI. The IL-1β and TNF-α expressions increase in the cortex within the first few hours in the rodent models of TBI before returning to baseline levels by 24 h [in vivo rodent models, the expression of chemokines and PTGS2 increases within hours of cortical injury and, unlike IL-1β and TNF-α, remains at elevated levels at 24 h or beyond [ex vivo slice system has negligible infiltration of leukocytes. The inflammatory gene expression in slices is regulated by a genetic background [Microglia are the resident CNS cells derived from monocyte/macrophage lineage and are the early responders to neural injuries. While microglia can be isolated or immortalized for use in highly valuable cell culture studies, one disadvantage to this system is that dissociated microglia are separated from their normal neural contacts. Microglial phenotype and function are regulated by the unique and complex cellular and extracellular signaling molecules . Studyin in vivo ,26, it i by 24 h . Similarr beyond -9. Remarckground  and can ckground -27. The in vivo rodent models and clinical studies in humans. Swine have gyrencephalic brains similar to humans in which 50% of the brain is comprised of white matter in contrast to the lissencephalic rodent brain that contains 15% white matter. Outbred swine, which are less expensive than primate species and physiologically similar to humans, are increasingly being used to study other trauma and other diseases involving the immune system such as organ transplant biology as they have fewer immunologic hurdles to overcome from swine to human [While cortical slices help fill the translational gap between cell culture and intact rodent models of injury, a gap in knowledge of acute cortical injury response also exists between to human . Swine ato human ,29 and bto human . Swine aThis report demonstrates the early transcriptional inflammatory response following cortical injury in piglets. In agreement with previous findings using outbred swine , which pin vivo changes reported with the rodent models of TBI. This slice injury approach can bridge the gap in experimental systems that exists between higher throughput cell culture and lower throughput animal models to facilitate immunotherapeutic discovery and the understanding of the acute immunological reaction to cortical injury. Later stages of preclinical development of immunomodulatory drugs would benefit from injury models using swine, which may more reliably model the brain anatomy and subject variability in human studies. The ex vivo mouse cortical slice injury system and the in vivo piglet model of brain injury can serve as additional preclinical systems to facilitate the discovery of therapeutic agents for TBI aimed at regulating early inflammatory mediator expression.New experimental systems to investigate the acute immunological reaction may facilitate the discovery of early interventional drugs to mitigate the disability and fatality after TBI. In the current study, two disparate mammalian systems of cortical injury illustrate an early increase in pro-inflammatory transcripts. The mouse cortical slice system reveals a temporal change in cytokine and chemokine expressions that is similar to the