Nanotechnology is the field which deals with fabrication of materials with dimensions in the nanometer range by manipulating atoms and molecules. Various synthesis routes exist for the one, two and three dimensional nanostructures. Recent advancements in nanotechnology have enabled the usage of block copolymers for the synthesis of such nanostructures. Block copolymers are versatile polymers with unique properties and come in many types and shapes. Their properties are highly dependent on the blocks of the copolymers, thus allowing easy tunability of its properties. This review briefly focusses on the use of block copolymers for synthesizing one-dimensional nanostructures especially nanowires, nanorods, nanoribbons and nanofibers. Template based, lithographic, and solution based approaches are common approaches in the synthesis of nanowires, nanorods, nanoribbons, and nanofibers. Synthesis of metal, metal oxides, metal oxalates, polymer, and graphene one dimensional nanostructures using block copolymers have been discussed as well. Nanotechnology is a relatively new field which involves control and exploitation of structures with sizes in the nanometer range. Goals in this field include fabrication of materials with properties that are novel and improved and are likely to impact different fields . NanotecTwo main approaches exist for the synthesis of 1, 2 and 3-dimensional nanostructures such as nanowires, nanotubes, and nano-masks. These are top-down and bottom-up approach  Fig. . AnionicBlock copolymers as compared to other copolymers have superior properties and these properties highly depend on the clustering and structural arrangement of the blocks. They allow the combination of significantly high melting points and elastic properties in the same polymer. Block copolymers also exhibit increased tensile strength, modulus, and elongation by suitable selection of the blocks. Other properties like moisture recapture, ability to absorb dyes also depend on the block copolymer’s structure . The phaBlock copolymers create well-ordered structures by undergoing microphase separation. Their properties can also be controlled through manipulating the three critical parameters mentioned above. Block copolymers can be used to synthesise different nanostructures due to their fitting size and shape. Their properties are also easily tunable by simply changing the monomer used, their compositions and molecular weight. Using block copolymers for fabricating nanostructures is simple and affordable as well . This seNanowires are one-dimensional nanostructure with a very high aspect ratio and a diameter in the nanometer range. Such a dimension allows nanowires to manipulate and control various subatomic particles like electrons, photons, phonons etc. to give rise to different applications –38. Base3O4) have been fabricated into 3-D crossing nanowires using soft chemistry templating synthesis. Here, Manganese chloride was filtered and mixed with block copolymers to form a sol of the same. The resultant sol was then aged overnight and a change in color was observed. This sol was then deposited on the silicon wafer which was then followed by calcination. These samples, were then characterized by SEM ] thin film. This was later annealed to synthesize molybdenum disulfide nanowires on SiO2/Si substrates. The experimental conditions were modified in such a way that they produced either polycrystalline or amorphous molybdenum disulfide nanowires. These were later characterized through TEM, Raman spectroscopy and X-Ray Photoelectron Spectroscopy (XPS) and XRD. It revealed that at higher annealing temperatures there was a transition from amorphous phase to polycrystalline phase with a decrease in excess elemental sulfur  using this technique for electrooxidation and sensing of ethanol. This was done by using the low molecular weight block copolymer to create a nanopattern which was transferred to the substrate through plasma etching. The orientation of the block copolymer was controlled through polymer brushes which were hydroxyl terminated. The silicon nanowires produced were characterized by SEM, TEM, and Energy Filtered Transmission Electron Microscopy (EFTEM). These studies revealed nanowires in sub-nm range with a high degree of domain alignment .Fig.Fig3N4 wb-poly(propylene oxide) (PEO-b-PPO) has been investigated on the morphological control of radial spherical ZnO nanorods. Solutions of different molar ratios of the block copolymer was added to the aqueous solution of Zn(C2H3O2)2·2H2O followed by addition of NaOH solution to the PEO-b-PPO modified zinc solution. SEM was used to further characterize the precipitate obtained to reveal ZnO nanorods. The length and the diameter of the hexagonal facet of each rod decreased with increase in copolymer concentrations. The effectiveness of photocatalytic degradation of the nanorods also increased with increase in their surface areas. However, there was no improvement in the antibacterial activity of the nanorods due to their sizes  was spin-coated on the mild plasma treated graphene substrate followed by annealing to achieve perpendicular domain orientation. The PMMA domain was then selectively removed through a CO2 based etching process and the pattern formed was subsequently transferred into the underlying graphene. The residual PS was removed by soaking it in THF, followed by drying of the sample at room temperature under vacuum. Characterization studies by AFM and SEM revealed successful removal of the PMMA block and SNT layers. Since graphene is placed between the block copolymer and SNT, this observation showed the successful etching of graphene thus transforming it into graphene nanoribbons. Also, the structure of graphene nanoribbons was identical to the PS fingerprinting pattern, thus confirming the pattern transfer from the block copolymer mask to fabricate the nanoribbons [In another study, FET characteristics and photoelectric properties of graphene nanoribbons having 9–12 nm ribbon widths that were fabricated through cylindrical PS-oribbons . Graphenoribbons .Nanofibers are defined as fibers with diameters less than 100 nm. They can be made from a variety of materials like carbon, semiconductors, polymers, and cellulose and find applications in tissue engineering, reinforcement in composites, filter media and micro/nano-electro-mechanical systems. Conventional synthesis techniques for nanofibers involve template based, electrospinning, nanoimprint lithography, and electrochemical approaches . DiblockSelf-assembled block copolymer nanofibers are excellent materials having applications as viscosifying agents in complex fluids or in biomedical field. The preparation of polymeric nanofibers is based on an aqueous emulsion polymerization using the RAFT method. RAFT agents were synthesized followed by the synthesis of the block copolymers using these RAFT agents under different pH and salt concentrations. The effect of these parameters on the formation of nanofibers was studied by TEM. With RAFT agents, at acidic pH or high salt concentrations, non-spherical morphologies were favored. The formation of the nanofibers also depended on the monomer concentration. With the conversion, the morphology of the nanofibers changed from spherical to worm-like due to increasing viscosity .Formation of nanofibers using block copolymers under cylindrical confinement has been reported as well. The two-fluid coaxial spinning technique was used to encapsulate the desired block copolymer as the core with another protective polymer as the shell. Advantages of the two-fluid electrospinning include (a) The shell fluid itself being electro-spinnable serves as a process aid for the block copolymers. Removal of these shells, results in ultrafine block copolymer fibers, (b) smaller diameter fibers are produced due to the independent control of the two fluids that permit wider changes in the diameter of the core fiber, (c) the integrity of the fiber does not get compromised due to the cylindrical confinement of the shell polymer which has a higher glass or melt transition temperature, thus creating a temperature window. TEM images confirm the continuous core–shell nature of the nanofibers. It also shows well-defined structures consisting of concentric layers because of curving of the lamellar phase due to fiber confinement .2O3 hybrid nanofibers have been prepared by processing films of the triblock copolymer polystyrene-block-poly(2-cinnamoyloxyethyl methacrylate)-block-poly(tert-butyl acrylate) (PS-b-PCEMA-b-PtBA). This preparation involved blocks in thin films getting self-assembled into concentric PCEMA and PtBA cylinders dispersed in a matrix of PS. These cylindrical structures were locked through by photocrosslinking the PCEMA shells. When these shells were dissolved in THF, nanofibers were produced with PtBA cores, PCEMA middle layers, and PS coronas. TEM images revealed PtBA cores that are 20 nm in diameter. Fe2O3 loading in the caused the nanofiber dispersion to become red. These nanofibers had Fe2O3 content to be about 0.28 g. TEM images of Fe2O3 nanofibers revealed fibers around 20 nm, which as the same as the polymer nanofiber [Polymer/Feanofiber .Nanotechnology has seen many advances in recent years and several synthesis techniques for nanostructures have been developed. These techniques can be mainly classified into top down and bottom-up approaches. Block copolymers have two or more blocks arranged in a defined manner. This arrangement is dependent on factors like Flory–Huggins interaction parameter, degree of polymerization, architectural constraints, and composition. By varying these parameters, it is possible to control the phase behavior and morphology of block copolymers. Such a flexibility enables block copolymers to be used for fabrication of nanostructures as a template or in a solution form. They can also be used to lithographically fabricate nanostructures. One dimensional nanostructures like nanowires, nanorods, nanoribbons, and nanofibers have unique electronic, chemical, optical, magnetic, and mechanical properties due to their nanostructure which enable them to have several applications in different fields of Science. Synthesis of such one-dimensional nanostructures using block copolymers has been discussed in this review. The techniques discussed show the versatility and effortlessness of block copolymers to synthesis these nanostructures.


Dear Editor,We report the case of a 60-year-old female patient who reported a one-week history ofpain in the left hypochondrium, fever, vomiting, and diarrhea. The physical examinationand laboratory tests showed no significant changes. Ultrasound showed a septated cysticmass, alongside the stomach, with thick walls and containing debris, although withoutany vascularity seen on the Doppler flow study . For clathat is extremely rare,3. It was first described in 1969 byKnoblich, who characterized it as lung injury. Lewars described the first oral lesion in 1971, the initial reports of the disease inthe extraoral gastrointestinal tract not appearing until 2001.Pulse granuloma is a benign lesion, typicallyindigestible cellulose deposited under the mucosa. Most pulse granuloma patients have a history of bowel disease,including diverticulitis, fistula, perforation, ulcerative colitis, appendicitis, oranastomotic leakage, allowing theforeign body to reach the deep layers of the intestinal wall. The oral cavity is thesite most often affected, the occurrence of pulse granuloma at other sites beingextremely rare. However, there havebeen reports of pulse granuloma in the stomach, small intestine, colon, peritoneum,mesentery, genitourinary tract, and skin,7.Pulse granuloma is characterized by a chronic granulomatous reaction to a foreign body ofvegetable origin, of a broad range of ages, cases having been described in patientsfrom 13 to 85 years of age. Thesymptoms are vague and nonspecific,occasionally including abdominal pain and discomfort.The physical examination is usually unremarkable, although apalpable mass can be identified,7.Pulse granuloma predominantly affects males. Nevertheless, because foreign bodies of vegetable origin do notproduce hyperintense images, the diagnosis is not usually obtained by CT. Uppergastrointestinal endoscopy is a useful tool in the study of gastric lesions and allowsthe collection of material for histopathological evaluation. However, endoscopicbiopsies are usually small and superficial, which can make it difficult to confirm thediagnosis of granuloma pulse. Thediagnosis is made through exclusion on the basis of the histopathologicalfindings. The possibility ofpulse granuloma should be considered in cases of expansive lesions in thegastrointestinal tract, the maindifferential diagnoses being adenocarcinoma, gastrointestinal stromal tumor, andleiomyoma. The definitivetreatment is surgical intervention.The imaging evaluation of pulse granuloma is usually made either by ultrasound, thefindings of which are often nonspecific, or by CT, which is more relevant because of itshigh sensitivity and specificity for the detection and characterization of foreignbodies in the gastrointestinal tract


Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens.Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major  Neisseria  that have been used as experimental vaccine adjuvants. Such activities reflect their capacity to activate innate immune responses through pattern recognition receptors, such as toll-like receptors (TLRs) . In addieisseria , Haemophmophilus , Pasteurteurella , Fusobacacterium , ShigellShigella , 10, andlmonella –14 on thlmonella , 13–15. ligation .Salmonella Typhi expresses multiple porins  and B cells  (a model antigen), an inactivated H1N1 2009 pandemic influenza virus, and a Vi polysaccharide, T-independent vaccine. These data show that porins promoted responses to all three antigens, indicating that these proteins may be used as adjuvants to a range of different types of antigens.Here, we evaluated the potential of the major All animal procedures were conducted in accordance with national guidelines , following review and approval by the Specialties Hospital Ethics Committee of Instituto Mexicano del Seguro Social (IMSS) (project number CNIC 2006-785-076).Endotoxin-free OVA was purchased from Seikagaku Corp. . Pandemic influenza virus (strain A/Mexico/4482/2009(H1N1)) was grown in chicken embryos and kindly provided by the Instituto Nacional de Diagnóstico y Referencia Epidemiológicos (InDRE), Mexico City. The virus was inactivated using formalin [inactivated influenza A virus (iIAV)] and quantified by hemagglutination. For ELISA assays, iIAV was concentrated with 6% polyethylene glycol 8000  and further purified by centrifugation in a 20–60% sucrose density-gradient in NTE Buffer  as described elsewhere , and proS. Typhi ATCC 9993 as previously described , and all batches tested negative . Digested porins (PorK) were prepared with 10 μg of proteinase K  per 30 μg of porins, followed by overnight incubation at 37°C and inactivation of the enzyme at 70°C for 1 h. LPS from Escherichia coli 0111:B4 was purchased from Sigma . Rehydragel was used as an alum control . Typhim Vi vaccine was obtained from Sanofi Pasteur .Porins were purified from escribed , 27. LPS323–339 transgenic mice were bred at the animal facilities of the Experimental Medicine Department, Faculty of Medicine, Universidad Nacional Autónoma de México (UNAM).Male BALB/c mice (6- to 8-week olds) were purchased from Harlan Laboratories . DO.11.10 mice OVAS. Typhi porins, 10 μg of proteinase K digested porins, 5 μg of LPS, or 100 μg alum. Animals were boosted on day 15, and blood samples were taken at the indicated time points. For Vi CPS experiments, mice were immunized i.p. with 10 μg of Typhim Vi CPS vaccine alone or with 10 μg of S. Typhi porins, or proteinase K digested porins, with a boost on day 15.BALB/c mice were immunized i.p. with 100 μg of OVA, 4 hemagglutinating units (HAU) of iIAV alone or with 10 μg of major 6 CFSE-labeled CD4+ T cells from DO.11.10 mice were adoptively transferred i.v. After 24 h, mice were immunized s.c. in the footpads. Three days after immunization, the popliteal lymph nodes were extracted and CD4+ T cells were purified by negative selection with Dynabeads® . To assess in vivo T-cell proliferation, CD4+ T cells were stained with PE-conjugated KJ1-26 mAb (against DO.11.10 transgenic TCR) and APC-conjugated anti-CD4 . To determine the cytokine production, CD4+ T cells were cocultured at a ratio of 3:1 with splenic DCs purified by positive selection from naïve mice. DCs were used alone or pulsed with 100 μg of OVA. Then, 24 h after stimulation, the supernatants were collected, and the cytokines were quantified using a Th1/Th2/Th17 CBA kit following the manufacturer’s instructions . Data were acquired on a FACSCalibur  and analyzed using FlowJo 7.5 software .A total of 5 × 102O2 . The reaction was stopped with 1.25 M H2SO4, and the optical densities were read at 492 nm using an automatic ELISA plate reader . The cutoff value was defined as threefold above the mean values of the negative controls.High-binding, 96-well polystyrene flat bottom plates  were coated with 15 μg of OVA , 1 μg of iIAV or 1 μg of Typhim Vi vaccine per well, each dissolved in 0.1 M carbonate buffer (pH 9.5). Non-specific binding was blocked with 5% non-fat dry milk diluted in PBS pH 7.2. Sera were serially diluted twofold and incubated for 1 h at 37°C. Peroxidase-conjugated anti-mouse IgM, IgG H + L, IgG2a, IgG2b, or IgG3  were diluted at a ratio of 1:1,000. The plates were developed with 0.5 mg/mL ortho-phenylenediamine  in 0.1 M citrate buffer (pH 5.6) containing 0.08% HHigh-avidity IgG antibodies were measured including a wash with a mild-denaturing agent to discriminate low-avidity antibodies, which are more likely to dissociate from the antigen–antibody complexes . BrieflySera were treated with receptor destroying enzyme  for 19 h at 37°C, according to the manufacturer’s instructions. Sera were serially diluted twofold in PBS using V-bottom plates . Diluted sera were incubated 30 min at RT with 8 HAU/25 μL of a pandemic influenza virus strain A/México/4482/2009 (H1N1). After incubation, 0.5% of chicken red blood cells were added to the plates and incubated 30 min at RT. The hemagglutination inhibition titer was established as the highest dilution of sera where hemagglutination was completely inhibited.P-values < 0.05 were considered significant. Significant differences are depicted as *P < 0.05, **P < 0.01, and ***P < 0.001.Statistical analysis was performed with GraphPad Prism 6.0  applying one-way analysis of variance test with Bonferroni’s multiple comparison correction. S. Typhi porins, hereafter referred to as porins, had an adjuvant effect on OVA antibody responses, we immunized BALB/c mice with OVA, OVA + porins, or OVA + proteinase K digested porins (PorK). Additionally, we included two groups immunized with OVA + alum and OVA + LPS to compare the relative effects of major S. Typhi porins to other known adjuvants. OVA-specific antibody titers were measured at different time points after immunization. OVA + porins induced higher IgG antibody titers, which remained detectable up to 120 days after immunization  virus (iIAV) plus S. Typhi porins, and we measured the antibody responses against influenza virus. We found that the addition of porins increased IgG antibody titers to iIAV, to a similar extent as alum or LPS, and that the response was maintained up to 120 days post-immunization , such as  in mice . Vaccina in mice . Current in mice . The Vi  in mice . To over in mice , 53.Salmonella Typhi porins could enhance antibody responses to the purified, unconjugated Vi CPS, reflecting a similar finding when minor porins are used . The activation of these receptors by porins might mediate the activation of APCs, such as DCs and macrophages. Activation of APCs then would induce the expression of costimulatory molecules and class II MHC expression and increase antigen presentation, as has been observed in DCs after stimulation or injection with porins (The mechanism mediating the adjuvant effect might be related to the agonist effect of h porins , 14. It h porins .S. Typhi porins displayed two patterns. The first pattern indicated that the magnitude and diversity of the response were augmented similar to the model antigen OVA. The second pattern indicated that the diversity of the response elicited by the antigen alone remained unchanged but the magnitude of the response was augmented. This finding could mean that the mechanism underlying the adjuvant effect of porins might vary depending on the antigen that is co-immunized. This possibility should be explored further.Overall, our observations revealed that the adjuvant effect of major The adjuvant effect of porins is similar when compared to LPS; however, LPS cannot be used as adjuvant in animals and human vaccines due to its high toxicity. Contrary to LPS, we have previously shown that porins immunization is safe and well tolerated both for mice and humans , 27, 28;S. Typhi porins OmpC and OmpF have adjuvant properties and potentiate, diversify, and extend B-cell responses to a diverse repertoire of antigens. They function in a manner associated with a Th1/Th17 profile. Our findings suggest that S. Typhi porins represent an additional choice of vaccine adjuvant for T-dependent and T-independent antigens.In conclusion, our results show that major MP-T performed the experiments, analyzed the results, and wrote the paper; NV-P and RP-P analyzed the results and wrote the paper; CG-C, CP-S, and MM-E designed the experiments and analyzed the results; IB and SP-T provided the reagents; LA-P, AC, and AI analyzed the results and revised the manuscript; and LB and CL-M designed the study, supervised the experiments, and revised the manuscript.Salmonella Typhi OmpC and OmpF porins being patented to use them as adjuvants for vaccines.The results of this work have led to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Robust topological edge modes may evolve into complex-frequency modes when a physical system becomes non-Hermitian. We show that, while having negligible forward optical extinction cross section, a conjugate pair of such complex topological edge modes in a non-Hermitian  Parity-time , hy(k), and hz(k) are periodic real-valued functions with period of 2π/d in k, and they form a x, y, and z refer to the directions in the k varies from −π/d to π/d. This closed loop is associated with a winding number which determines the Zak geometric phase of a band.It should be noted that The Zak phase of each band (+ or −) is usually expressed as18202136373839Ek± are real for all k ∈   winding number of k) about the hz axis as k varies. It can also be shown that all even (odd) integer values of w are equivalent and Zak phase is thus usually quantized as zero or π. Since there exist different approaches in defining the Zak phase, we also provide a comparison to the results in bi-orthonormal basis T, while the matrix Ak is related to dipole couplings and the polarizabilties. Eigenvalue ε1 is also the dielectric function of the plasmonic core, and is mapped to the plasmon frequency ω via the Drude model204549204549ωp and 1/τ are the plasma frequency and electron collision frequency for the plasmonic core.in which τ = 0). A detailed discussion on symmetry operators is given in the Methods.The finite plasmonic particle array has no x component) frequencies of a finite τ = 0 are shown in ω are real. This implies all the eigenvalues ε1 are real, so the entire bulk dispersion relation is in unbroken ω/ωp) = ±0.002. The existence of these complex edge modes are due to γ = π, which is classified as the  regime III. The evaluation of Zak phase for different gain/loss parameters are provided in The longitudinal mode :κ = (3/d)ln[s/(d − s)], ω0 is the complex frequency of an edge mode, and pn;σ(t) represents the time-domain dipole moments in the nth unit cell. Both of the edge modes decay spatially, and the mode patterns at point D are shown in where x-polarized electrical plane wave with magnitude E0 and frequency ω, as shown in N nanoparticles p = T are linearly dependent on the external wave E0 = T (see Methods):S(ε1) is a square matrix depending on ε1, and explicit expression is shown in where In 5859k0 = ω/c, c, and ε0 are, respectively, the photon wavenumber, the speed of light, and the permittivity in free space.where ω = 0.529ωp is due to the conjugate pair of edge modes, which drops drastically when Im(ε3) slightly increases from 0 . To obtain an eigenvalue problem with ε1 as the eigenvalue, we group terms with ε1. 1Nkpk = Mkpk, in which pk = T,where andMultiplying the matrix equation by whereGk+2π/d;AB = Gk;AB, we can write Ak = fkI2 + Hk, where I2 is a 2 × 2 identity matrix.and ith nanoparticle has the general form ω is the angular frequency and pi(ω) is the Fourier component. Defining the time reverse operator by We define the time-reversal operator From the above, A and B.For the case that the plasmonic cores are lossless, time reverse operation turns the energy gaining dielectric into energy losing dielectric and vice versa, which means the time reversed particle array is effectively the same as the particle array obtained by swapping the positions of nanoparticles S(ε1) in The analysis of N × 2N matrix A commutes with A−1 commutes with AA−1 = IN2, we have A commutes with A−1 from the left, we haveFirstly, if a general 2A and B commute with AB, also commutes with Secondly, if matrixes N, M, C3, and C4 commute with It is obvious to see from their definitions, ε1 is real, we have N−1M − ε1IN2 commutes with 1C3 + C4 commutes with 1 is real. As a result, again by S(ε1) below S(ε1) commutes with 1 is real.We see if kd = ±π + iκd20The analytical solutions of the topological edge modes are based on coupled-dipole method in quasi-static approximation. For simplicity, we consider a semi-infinite plasmonic particle array with only one edge on the left. The protected edge modes are required to satisfy both the boundary condition and the dispersion relation with complex wave vectors By assuming the dipole moments are in the Bloch’s form with complex wave vectors, we haveσ = A or B. The dipole moments in the first two unit cells have to follow the coupled dipole equation at the boundary:where pσ0; = 0 in pA2; = −pA1;eκd to pB1;/pA1;, we havewhich is simply obtained by putting pk;σ in k is real, it gives the dispersion relation; when kd = ± π + κd, it becomeswhich is a condition obtained by considering the boundary. On the other hand, the existence of the nontrivial solution which is another condition for the edge state.Eliminating κ ≥ 0), there is no solution for κ unless s > 0.5d, which is the case with non-trivial Zak phase. By putting pA1; = pB1; = 0 in 1 = −c2/c1, pA1; = 1, and pB1; = 0. Putting back to the Drude model As only decaying dipole moments are physical (eκd− = s3/(d − s)3 with κ < 0, and which is the left edge state resonant frequency. Solution for the particle array with a right edge can be obtained similarly. In this case, In our multiple scattering calculations, electromagnetic fields that satisfy the source free macroscopic Maxwell’s equations with the constitutive relations are expanded in series of vector spherical harmonics for each homogeneous region60β(k) is a real periodic function that β(k + 2π/d) = β(k) + 2lπ with integer l, and is known as the gauge freedom38π.There is a phase ambiguity on choosing eigenvectors in ω − ω0) behaviour5There have been ways to realize the gain medium, for example, the use of semiconductor quantum dots6666How to cite this article: Ling, C. W. et al. Anomalous Light Scattering by Topological Sci. Rep.6, 38049; doi: 10.1038/srep38049 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Tropomyosin receptor kinase B (TrkB) activation, rather than glutamate receptor alteration, was consistently found after the different manipulations. Immunohistochemistry further revealed peri-infarct neuronal TrkB activation and microvasculature augmentation in AUDA-treated and sEH KO mice, suggesting parallel neurovascular enhancement. Mechanistically, pretreatment with a selective TrkB antagonist ANA12 countered the effect of iLTP attenuation induced by sEH deletion ex vivo and abolished the infarct reduction in vivo. Together, the neuroprotective effects of sEH inhibition and gene deletion can both be mediated partially via enhancement of TrkB signaling which attenuated post-ischemic neuroexcitation and neurological deficits.Inhibition and deletion of soluble epoxide hydrolase (sEH) has been suggested to ameliorate infarction in experimental ischemic stroke possibly via vasoactive epoxyeicosatrienoic acids. However, it is unknown whether the neuroprotective mechanisms involve alteration of post-ischemic neuronal transmission and neurotrophic signaling. We used a permanent middle cerebral artery occlusion (MCAO) model in adult wild-type mice with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) post-treatment and in sEH knockout (sEH KO) mice. We found that sensorimotor recovery was significantly enhanced after MCAO in both AUDA-treated and sEH KO mice, with decreased sEH activity and brain infarction. Decreased post-ischemic long-term potentiation (iLTP) was observed in an  EETs and related epoxide fatty acids are signaling fatty acids generated from arachidonic acid through the cytochrome P450 epoxygenase pathway and have been shown to have protective effects on vasodilation5, angiogenesis6, as well as anti-inflammation10. It has been shown that sEH expression in the mammalian brain occurs mainly in astrocytes and vascular endothelial and smooth muscle cells as well as in a few neuronal populations in the striatum, cortex, and hippocampus14. Following middle cerebral artery occlusion (MCAO) in adult rodents, cytochrome P450 enzymes and arachidonic acid metabolites have been suggested to play essential roles in regulating cerebral blood flow and inflammatory responses to ischemia. Systemic administration of 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), an sEH inhibitor, has been shown to reduce infarct size and ameliorate functional deficits; these effects were attributed, at least partially, to increased EET levels and enhanced cerebral blood flow16. Similarly, sEH knockout (sEH KO) mice were also protected from ischemic stroke. Compared to wild-type (WT) controls, sEH KO mice had smaller infarcts in association with increased EET levels and collateral blood flow12, In contrast, transgenic mice with endothelial-specific overexpression of human sEH exhibited impaired vasodilatation and enlarged infarcts after MCAO in female but not in male mice17, suggesting that sEH may serve as a vasoactive modulator and a potential therapeutic target in ischemic stroke. However, the functional relevance of neuroglial sEH expression, as well as sEH blockade-mediated neuroprotective mechanisms against stroke, remain elusive.Soluble epoxide hydrolase (sEH), which is encoded by the EPHX2 gene, is found in nearly all mammalian tissues18. The infarct reduction was countered by intracerebroventricular injection of a potent but non-selective tyrosine receptor kinase inhibitor, K252a, which acts on tropomyosin receptor kinase (Trk)A, TrkB or TrkC. Moreover, exogenous 14,15-EET treatment in cultured murine astrocytes and a human astroglioma cell line subjected to oxygen-glucose deprivation (OGD) increased BDNF expression and cell viability, indicating that 14,15-EET-mediated production of BDNF by astrocytes and this effect might protect against ischemic injury in sEH KO mice18. In another study that used lipopolysaccharide-induced inflammation and repeated social defeat stress models, pretreatment with an sEH inhibitor and sEH KO were found to prevent the onset of depression-like behaviors and to increase BDNF-TrkB expression in the prefrontal cortex and the hippocampus, suggesting that BDNF-TrkB signaling confers the stress resilience in association with sEH blockade19. Nevertheless, these interesting findings have not elucidated how sEH blockade alters neural excitability after ischemic injury and whether BDNF-TrkB signaling mediates such neuroprotective effects. BDNF is a paracrine neurotrophic factor20 that is secreted by almost all cell types in the brain. BDNF binds to the high-affinity transmembrane receptor TrkB and the low-affinity p75 neurotrophin receptor to mediate various neuroplastic and neuroprotective effects throughout life22. Here, we investigate how pharmacological inhibition and gene deletion of sEH exerts neuroexcitatory modulation and neuroprotection against ischemic hypoxic brain injury by utilizing N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl) amino] carbonyl] phenyl]-benzo[b] thiophene-2-carboxamide (ANA12), a selective TrkB inhibitor23, and focusing on the role of BDNF-TrkB signaling and glutamate receptors. To do so, we adopted a permanent mouse MCAO model in vivo and post-ischemic long-term potentiation (iLTP) measurements in ex vivo hippocampal slices subjected to OGD. Given the potential neurovascular benefits of sEH inhibitors in preclinical stroke models and their safety profiles in humans25, our findings indicate that TrkB activation is involved in sEH blockade-mediated neuroprotection in acute ischemic stroke and open new possibilities for combination treatments with neuroprotectants and reperfusion by thrombolytic and/or endovascular thrombectomy treatments.A recent study reported that sEH KO mice subjected to MCAO had decreased infarcts in association with elevated brain-derived neurotrophic factor (BDNF) expression predominantly in peri-infarct glial fibrillary acidic protein (GFAP)-positive astrocytes26, we found that both the AUDA-treated mice and soluble epoxide hydrolase knockout (sEH KO) mice exhibited a significant reduction of infarct volume relative to the vehicle-treated and wild type (WT) mice  and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor types30. During the 5 minutes of OGD induction, there were no obvious differences in field excitatory postsynaptic potential (fEPSP) amplitudes among the acute hippocampal slices from WT, WT/Vehicle, sEH KO and AUDA-treated (n = 6/group) mice -induced post-ischemic long-term potentiation (iLTP) has been attributed to abnormal Caice Fig. , indicatice Fig. , suggest2+-impermeable AMPAR subunit GluA2. AUDA treatment upregulated the level of the NMDAR GluN2A subunit but did not have a significant effect on GluN2B or GluA2 subunit after MCAO  signaling, which induces NMDAR trafficking and activation. Compared to vehicle treatment, AUDA treatment upregulated mature BDNF (17 kDa) and phosphorylated TrkB (p-TrkB) . Pro-BDNF (35 kDa) and p75NTR (70 kDa) levels did not differ between groups Fig. . On the 36. Nevertheless, other studies of heart and kidney injury reported that in contrast to pharmacological inhibition, sEH gene deletion aggravated myocardial fibrosis and renal tubular inflammation in association with elevated HETEs, which might have an effect opposite to that of EETs on vascular regulation and inflammation28. However, such pro-inflammatory effects of HETEs were not observed here in sEH KO before or after stroke. The cerebral microvasculature of sEH KO was more abundant than that of WT mice regardless of elevated 20-HETE levels. This may be due to complex substrate shifting in transgenic mice after generations of adaptation to the changes caused by lack of sEH. Collectively, these findings suggest that increased p-TrkB in neurons, as well as enhanced collateral microvasculature and reduced inflammatory gliosis, may be associated with the neuroprotection mechanisms by sEH blockade. The student’s t-test was used to determine the significance of differences in quantitative immunoreactivities.Pharmacological inhibition of sEH significantly increased CD31-positive endothelial cells in the peri-infarct region, while increased CD31-positive cells were observed before and after MCAO in sEH KO mice Fig. . Additioin vivo 48 hours after MCAO  and genetic deletion of sEH significantly ameliorated ischemic infarction and sensorimotor deficits in association with overexpression of brain p-TrkB and NMDA-type glutamate receptors in adult mice. Moreover, neuronal expression of p-TrkB, as well as collateral microvasculature, were significantly enhanced in the peri-infarct cortex, while astrogliosis and microglial activation were reduced. We demonstrated, for the first time, that both the inhibition and deletion of sEH reduced post-ischemic neuronal hyperexcitability, i.e., iLTP, in an ex vivo. These enhancements were associated with the overexpression of postsynaptic NMDAR and AMPAR subunitsas well as extracellular signal-regulated kinase (ERK) phosphorylation37. The activation of ERK signaling mediates neuronal responses to neurotrophic factors and is required for NMDA-dependent LTP38. By contrast, here we demonstrate that both sEH inhibition and gene deletion decreased iLTP following OGD insult using extracellular recordings in the hippocampus. These decreases were associated with the overexpression of p-TrkB; however, the upregulation of NMDAR and AMPAR subunits was inconsistent across the models. The addition of the selective TrkB inhibitor ANA12 reversed the attenuation of iLTP ex vivo and the infarct size reduction in vivo. Hence, the neuroprotective effect of sEH inhibition may be mediated by TrkB activation rather than through glutamate receptors. It has been well documented that BDNF-TrkB activation enhances synaptic transmission and plasticity40. Recently, it has been suggested that sEH deletion and 14,15-EETs promoted astrocyte-derived BDNF production and exerted neuroprotective effects during OGD injury through BDNF-TrkB-ERK activation in vitro18. Moreover, pharmacological inhibition and genetic deletion of sEH have been shown to prevent the onset of depression-like behaviors induced by repeated experimental social defeat stress in association with upregulated BDNF-TrkB expression in the prefrontal cortex and hippocampus19. Our findings suggest that sEH blockade may not only enhance physiological LTP but also attenuate OGD-induced iLTP. The attenuation of iLTP and infarction against ischemic injury by pharmacological inhibition and genetic deletion of sEH involves, at least in part, TrkB activation. The neural mechanisms can be further elucidated using forebrain-specific42, glia-specific or neuron-specific43 TrkB knockout mice.The impact of sEH on synaptic plasticity is largely unknown. We previously reported that sEH inhibition (AUDA) enhanced neuronal synaptic transmission and physiological long-term potentiation (LTP) following high-frequency stimulation using whole-cell and extracellular recordings in the mouse prefrontal cortex 26. It is unknown whether the neuroprotective effects of iLTP attenuation and p-TrkB overexpression might be EET-dependent. Second, we could not determine which glutamate receptor might mediate iLTP modulation via sEH blockade using extracellular fEPSPs. Whole-cell patch clamp recordings or synaptoneurosome isolation would be required to make this distinction44. The mechanisms underlying the effects of sEH on synaptic plasticity remain unclear. Third, we can not exclude possible vascular mechanisms by sEH blockade because AUDA treatment, but not sEH KO, lowered blood pressure after MCAO. Nevertheless, we did not observe significant changes in peri-infarct blood flow by sEH blockade. Fourth, we used an ex vivo hippocampal OGD model for convenience of iLTP recording, which is not supplied by MCA, therefore not accurately reflective of an in vivo MCAO model. Fifth, BDNF mRNA and protein are present at extremely low levels in the brain20, and after ischemic injury, brain BDNF expression increases in a transient manner and then dramatically decreases45. Consistent with TrkB overexpression already before MCAO, sEH gene deletion did not significantly increase BDNF protein levels after MCAO in the current study. Additionally, we could not completely rule out that neurotrophin4-TrkB signaling also plays a role in TrkB activation. Finally, the pharmacokinetics of ANA12 in vivo limit its effects to 4 hours23; more frequent administration of ANA12 may be feasible for TrkB inhibition.There are limitations to our study, and further studies are needed. First, we did not have direct 14,15-EET measurement data because a liquid chromatograph-mass spectrometer was not available, and the manufacturer of the ELISA kit stopped production before we could finish our experiments. According to previous publications, sEH blockade likely increases EET levels in the brainex vivo, and this effect was reversed by selective TrkB inhibition. sEH genetic polymorphisms may increase or decrease sEH activity46, which might affect TrkB activity. Our findings provide novel insights into the TrkB-mediated neuroprotection and ischemic resiliance conferred by pharmacological inhibition and gene deletion of sEH.In conclusion, we demonstrated the protective potential of sEH inhibition after acute ischemic stroke in amelioration of functional deficits and provided evidence that this protection involves TrkB activation. Pharmacological inhibition and gene deletion of sEH attenuated iLTP following OGD insult in the hippocampus The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.Ephx2tm1Gonz/J, The Jackson Laboratory, n = 53) were anesthetized with intraperitoneally administered chloral hydrate  and received permanent middle cerebral artery occlusion (MCAO) surgery using a procedure described previously47. Briefly, the dominant limb was determined based on the skilled forelimb reaching test (see below). Subsequently, the distal MCA contralateral to the dominant limb was exposed by craniotomy and directly cauterized, followed by simultaneous occlusion of the bilateral common carotid arteries using microclips for 20 minutes to paralyze the dominant forelimb. The body temperature was maintained with a homeothermic heating pad with rectal temperature control at 37 ± 0.5 °C. For the pharmacological inhibition of sEH, 61 WT mice were randomly allocated to receive a daily intraperitoneal injection of either vehicle (2% dimethyl sulfoxide (DMSO), Sigma-Aldrich, n = 40) or AUDA48  for 7 consecutive days post-MCAO. The animal experiments were approved by the Institutional Animal Care and Use Committee at the Taipei Veterans General Hospital. All methods were performed in accordance with the relevant guidelines and regulations.Adult male C57BL/6 J WT mice  and sEH KO mice 51 and the adhesive removal test52 . For the rotarod test, the mice were pre-trained for 5 days before surgery. Within each session, the rotarod speed was increased from 4 to 40 rpm with an acceleration rate of 8 rpm/min. The mice performed 3 times of 10-minute test sessions of running. The latency to fall from the rotarod was recorded and averaged. For the skilled forelimb reaching task, i.e., the pasta matrix reaching task, the mice were pre-trained for 2 weeks to learn to break and retrieve pieces of uncooked capellini from a row of vertically oriented pieces of pasta (matrix) until they achieved a maximum of 30 pieces in 30 minutes. The mouse was placed in an acrylic chamber (15 × 8.5 × 20 cm) containing a slit (13 × 0.5 cm at the center of the 15 cm long side). The capellini matrix was then placed in front of the slit. The number of pieces of pasta removed in 30 minutes were recorded for analysis. For the adhesive removal test, a forelimb was cleaned, and a piece of adhesive tape (0.3 × 0.4 cm) was attached to the limb. The timer started once the mice were placed into a cylinder. The time at which the mouse began to remove the tape and the time at which the tape was completely removed was recorded.Before MCAO and 1 to 7 days after, blind observers longitudinally measured performance on the rotarod test3) was calculated as the sum of the infarct areas on each slice53. Both the surgeon and analyst were blind to the experimental groups.After 2 or 7 days of MCAO and behavioral measurements, the mice were decapitated after intraperitoneal injection of urethane (1–1.5 g/kg). Immediately after, fresh brains were harvested for quantification of infarct size, sEH activity, fatty acids, and proteins. To quantify the infarct, we sectioned the brain into standard 1 mm coronal slices using a brain matrix slicer , stained the brain slices with a 2% solution of the vital dye 2,3,5-triphenyltetrazolium chloride  at 37 °C for 30 minutes in the dark and fixed them with 10% formalin at room temperature overnight . The area of the infarct was delineated and measured using AIS software (Imaging Research Inc.), and the total volume  kits immediately after the tissue was homogenized according to the manufacturer’s instructions (Detroit R&D). For DHET ELISA, the homogenate was acidified with acetic acid, extracted with an equal amount of ethyl acetate several times and then dried using a vacuum concentrator (ThermoFisher). The residue was dissolved in ethanol and diluted with dilution buffer for the ELISA.To measure sEH activity and fatty acids, the ipsilesional hemisphere was isolated, weighed and homogenized with additional T-PER Tissue Protein Extraction  (n = 5/group). The hydrolase activity of sEH was determined using a cell-based assay kit 55.For western blotting, the aforementioned fresh homogenate that was prepared in T-PER Tissue Protein Extraction buffer (ThermoFisher) was mixed with 0.5% protease inhibitor (Sigma-Aldrich) and 0.5% phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich) and centrifuged at 10000xg for 20 minutes to obtain supernatants (n = 8/group). The protein concentration of the supernatants was determined using a NanoVue Plus Spectrophotometer. Equal amounts of protein (40 µg) were loaded onto 7.5%, 12% or 15% gels depending on the target protein size. The proteins were then separated using SDS-PAGE, transferred onto polyvinylidene difluoride membranes and blocked with 5% skim milk powder (Fluka) for 60 minutes. The following primary antibodies were then used for incubation overnight at 4 °C: rabbit anti-BDNF , rabbit anti-phosphorylated TrkB , rabbit anti-GluN2A , rabbit anti-GluN2B , rabbit anti-p75NTR , rabbit anti-GluA2  and rabbit anti-β-Actin . The membranes were then incubated with secondary HRP-conjugated antibodies  for 1 hour at room temperature. The immunoreactivity was detected with Clarity Western ECL Substrate (Bio-Rad). X-ray films (Fuji) were exposed for the different duration to ensure optimal densities. Relative optical densities were normalized to the levels of the internal control β-actin and then measured as fold changes using ImageJ55.After 2 or 7 days of MCAO, the mouse was anesthetized and transcardially perfused with 20 ml normal saline containing 10 U/ml heparin followed by 20 ml 4% paraformaldehyde in phosphate-buffered saline (PBS) . The brain was dissected and post-fixed in 4% paraformaldehyde overnight and then transferred to 30% sucrose for days. Afterward, 20 µm thickness frozen cryostat brain sections were attached to silane-coated glass slides. The slides were incubated in a blocking solution containing 3% donkey serum albumin (Abcam) and 0.3% Triton X-100 (Sigma-Aldrich) in TBS for 1 hour at room temperature and immunostained with primary antibodies including rabbit anti-CD31 , anti-p-TrkB , anti-NeuN , anti-GFAP , and anti-Iba1  overnight at 4 °C. The slides were then further incubated with fluorescence marker-conjugated secondary antibodies  for 2 hours at room temperature in the dark. After the slides were washed thoroughly with TBS, coverslips were sealed onto the glass slides with Vectashield Fluorescent Mounting Medium containing DAPI nuclear counterstain (Vector Laboratories). Images were acquired and averaged over a standardized sampling sites of 3 slices per mouse and 2–6 mice per group  using an Olympus FV1000i confocal laser scanning microscope. Quantification was performed with ImageJ2 and 5% CO2) artificial cerebrospinal fluid . The brain slices (400 µm thickness) were prepared as described previously56 and placed in a beaker of oxygenated ACSF at room temperature for at least 1 hour before recording. A single slice was transferred to a recording chamber consisting of a circular well (2 ml volume) and perfused constantly at a rate of 2–3 ml/min at 32 ± 1 °C. A bipolar stimulating electrode  was inserted into the Schaffer collaterals to deliver electrical stimuli . Field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum of the CA1 subregion with a glass pipette containing 1 M NaCl and a Multiclamp 700B amplifier. Baseline field potentials were recorded every 10 s for 10 minutes and adjusted to 30–40% of the maximal responses. For OGD and iLTP induction, glucose-containing ACSF gassed with 95% O2/5% CO2 was replaced with sucrose-containing ACSF gassed with 95% N2/5% CO2 for 5 minutes. Then, the perfusion solution was switched back to normal ACSF, and the slices were re-oxygenated with 95% O2/5% CO258. iLTP was continuously recorded for 60 minutes after OGD. The average fEPSP amplitude during the 5 minutes of OGD induction and the last 10 minutes of iLTP expression were compared across groups (n = 6 slices/group).Adult WT (n = 12) and sEH KO (n = 18) mice were decapitated, and their brains were immediately dissected and placed in ice-cold (4 °C) oxygen-saturated .To investigate whether TrkB mediated the effects of sEH inhibition and deletion on the attenuation of brain infarction and iLTP, we pre-treated the experimental groups with the selective TrkB inhibitor ANA12, before MCAO and OGD, respectively. WT and sEH KO mice were pretreated with intraperitoneal injection of ANA12  or vehicle  every 12 hours for 3 times before the MCAO surgery to ensure TrkB inhibition. For the p-value < 0.05 was considered statistically significant.We used SPSS (Windows version 22) for the data analyses. The repeated-measures analysis of variance test (ANOVA) was used to compare the behavioral data, with “time point” as the within-subject factor and “group” as the between-subjects factor. The ANOVA was followed by post-hoc Bonferroni tests at every time point to determine any group difference. We used independent Student’s t-tests or one-way ANOVA to evaluate group differences in infarct size, sEH activity, lipid metabolites, western blotting, immunohistochemistry and fEPSP amplitude measurements. A Supplementary Information


It innervates the mesencephalic locomotor region (MLR) from basal vertebrates to mammals. This pathway was shown to increase locomotor output in lampreys, and could very well play an important role in mammals. Here, we provide a detailed account on the newly found dopaminergic pathway in lamprey, salamander, rat, monkey, and human. In lampreys and salamanders, dopamine release in the MLR is associated with the activation of reticulospinal neurons that carry the locomotor command to the spinal cord. Dopamine release in the MLR potentiates locomotor movements through a D1-receptor mechanism in lampreys. In rats, stimulation of the substantia nigra pars compacta elicited dopamine release in the pedunculopontine nucleus, a known part of the MLR. In a monkey model of Parkinson's disease, a reduced dopaminergic innervation of the brainstem locomotor networks was reported. Dopaminergic fibers are also present in human pedunculopontine nucleus. We discuss the conserved locomotor role of this pathway from lamprey to mammals, and the hypothesis that this pathway could play a role in the locomotor deficits reported in Parkinson's disease.In vertebrates, dopamine neurons are classically known to modulate locomotion via their ascending projections to the basal ganglia that project to brainstem locomotor networks. An increased dopaminergic tone is associated with increase in locomotor activity. In pathological conditions where dopamine cells are lost, such as in Parkinson's disease, locomotor deficits are traditionally associated with the reduced ascending dopaminergic input to the basal ganglia. However, a descending dopaminergic pathway originating from the  Several lines of evidence indicate that this new dopaminergic pathway is functional in rats, and could also be present in humans. Future research should address whether the descending dopaminergic pathway potentiates locomotion in mammals as in basal vertebrates, whether it contributes to other PPN functions, and whether this dopaminergic innervation degenerates in PD patients.DR and RD wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this perspective, we highlight some of the recent advances in the development of molecular and biomolecular systems for performing logic operations and computing. We also present a blueprint of a chemical Turing machine using a processive catalytic approach. To comply with the rapidly increasing demand of information storage and processing, new strategies for computing are needed. The idea of molecular computing, where basic computations occur through molecular, supramolecular, or biomolecular approaches, rather than electronically, has long captivated researchers. The prospects of using molecules and (bio)macromolecules for computing is not without precedent. Nature is replete with examples where the handling and storing of data occurs with high efficiencies, low energy costs, and high-density information encoding. The design and assembly of computers that function according to the universal approaches of computing, such as those in a Turing machine, might be realized in a chemical way in the future; this is both fascinating and extremely challenging. In this perspective, we highlight molecular and (bio)macromolecular systems that have been designed and synthesized so far with the objective of using them for computing purposes. We also present a blueprint of a molecular Turing machine, which is based on a catalytic device that glides along a polymer tape and, while moving, prints binary information on this tape in the form of oxygen atoms. The resemblance of the Turing machine to naturally occurring molecular machineries that are capable of processing information, such as the ribosome and processive DNA (and RNA) polymerase enzymes, is striking and has recently motivated researchers to start programs on the development of biomolecular computers.4Essentially, a Turing machine works on an infinitely long tape and contains a head that can write and read symbols, while moving forward and backward on the tape. Although all modern computers obey the Turing machine concept, to date, no practically useful computing device that acts according to the original blueprint has been constructed.Herein, we describe recent developments in the construction of molecular and bio(macro)molecular systems for computing by focusing on information processing systems based on catalytic reaction routes that resemble information processing found in nature. We highlight catalytic enzyme approaches for developing logic gates, and the DNA-manipulating enzyme approach for information processing. Furthermore, we introduce the concept of processive catalysis as an important aspect of biological and future chemical Turing machines, and discuss our own efforts to construct such a synthetic molecular Turing device that works on a polymer tape encoded by inputs given by a tape head.2In the last seven decades, after the invention of the von Neumann-type computer architecture , enormou8Molecular computing gets its inspiration from biology, where information processing at the nanoscale is commonly found. Our brains, which carry out a myriad of information processing operations that rely heavily on chemical and electrical signals, are the best examples of natural, complex, molecular computer systems.Such captivating questions require renewed efforts in broad fields of science and paradigm shifts in the way we think about computer technologies. The majority of efforts to emulate the function of a brain involves the creation of artificial chemical analogues of neural-networks-like systems for computation.14In recent years, a great deal of effort has been made to mimic current silicon-based computing technology with the help of molecular systems. Some of these efforts are highlighted in the next section.2.1Modern computers are built on vast networks of transistor-based logic gates that execute binary arithmetic and Boolean logic operations. Logic gates are electronic switches, the output states (binary digits 0 or 1) of which are governed by the input conditions (binary digits 0 or 1); therefore, all information is encoded in a series of zeros and ones.16i.e., based on supramolecular interactions) that change between two distinctive states in response to external optical or chemical signals could be utilized as molecular switches, and hence be applied, at least in principle, to construct transistors for storage devices and molecular computing. The idea of information storage, with the help of molecular switches, was already proposed by Hirshberg back in 1956.A), which can be positioned at different locations through weak supramolecular interactions that can be regulated by an external stimulus, in this case acid.21Theoretically, individual molecules or self-assembled molecular systems , and four chemical inputs, namely, acetylcholine, butyrylcholine, O2, and glucose. The system can perform three logic gate operations in series, namely, OR, AND, and XOR .334e.g., all 154 sonnets of Shakespeare and the speech of Martin Luther King) could be stored in the form of DNA and retrieved with high, nearly 100%, precision.In nature, living cells function as universal Turing devices by reading, storing, and processing the myriad of information present in the intra- or extracellular environment. A prominent natural material present in cells that has been explored for molecular computing is DNA. DNA-based computing was first demonstrated by Adleman,A biomolecular copying nanomachine, such as the DNA polymerase enzyme system, which moves along a single strand of DNA, reading each base and writing its complementary base onto a new, growing strand of DNA, is one of the best natural examples of a classical Turing machine.i.e., short encoded double-stranded DNA molecules). The output is written on the same plasmid using DNA-manipulating enzymes as hardware. This advanced computing machine can algorithmically manipulate genetic codes.in vivo because they can interact directly with biochemical environment, offering potentially new approaches for gene therapy and cloning.Another recent development is the construction of the biomolecular equivalent of an electronic transducer. Transducers are electronic devices that can encode new information using their output for subsequent computing in an iterative way.Some weaknesses of DNA-based computing systems include the fact that they are fragile and also complex, that is, they make use of a quaternary  instead of a binary coding system.5Recent years have witnessed a large growth in the development of molecular machines based on interlocked molecules (catenanes and rotaxanes) and their application in the field of molecular electronics.The turnover number of this processive oxidation reaction is relatively high, as a result of the high effective molarity created because the catalyst remains attached to the polymeric substrate. This behavior is remarkably different from that of a distributive catalyst, which detaches itself from the substrate after every catalytic cycle.e.g., cis-alkene, trans-alkene, and pendant alkenei.e., 3 alkene and 3 epoxide functions). The required tape head  will be a chemically linked porphyrin double cage compound of the type depicted in i.e., a catenane) that can rotate and deliver commands exerted by external stimuli. The other cage would then be threaded onto the above mentioned polymer tape. The catenane compound can be synthesized by employing a ring-closing metathesis reaction with a Grubbs catalyst, as we have shown previously.e.g., light, electrons, or acid/base; see R or S) states, which can be attained by binding chiral guests  in the upper cavity of the double cage compound. This chiral information will be transferred to the other cage by allosteric modulation, that is, shape changes, which is, in principle, possible, as we have already shown for the binding and threading of (polymeric) guests in double cage porphyrin systems.A blueprint of such a system is presented in To become a universal Turing machine that can perform computations, many more criteria have to be met, even for simple machines with state/symbol pairs of , , and .6We have highlighted the ambition and possibilities of using molecular and bio-macromolecular systems for logic operations and computing. We also presented a catalytic approach, an operation denoted as “processive catalysis”, as a potential key element in the design of a future molecular Turing machine that would be completely synthetic. The field of molecular computing is rapidly developing, with new ideas coming from groups working in very different fields of science. This is opening the door for the design and construction of novel molecule-based computational devices. Although such future devices might execute operations similar to those of a Turing machine, one might ask if it could practically process information on a reasonable timescale. Further studies will have to answer this question, but it is evident that the development of molecular computers is an interesting and exciting direction of research in the bustling field of computer technology. Substantial progress can only be made if computer scientists, mathematicians, physicists, chemists, and biologist get together and work jointly on new, unorthodox programs.An interesting perspective, which has not yet received enough attention, is the application of Turing-like machines in medicine. In particular, DNA-based computing systems, which can read and transform genetic information, have the possibility to produce computational results in the form of genetic material that can directly interact with living organisms, which might prove to be useful in gene therapy, for example.Processive catalysis is one of nature's most frequently used tools whenever polymers are involved. For instance, almost all translational processes seen in the cell are processive, and proteins are literally born from processive catalysis.


Present results show that CNP did not affect either 3H-dopamine uptake in renal tissue or Na+, K+-ATPase activity; meanwhile, Ang-(1-7) was able to increase 3H-dopamine uptake and decreased Na+, K+-ATPase activity in renal cortex. Ang-(1-7) and dopamine together decreased further Na+, K+-ATPase activity showing an additive effect on the sodium pump. In addition, hydrocortisone reversed Ang-(1-7)-dopamine overinhibition on the enzyme, suggesting that this inhibition is closely related to Ang-(1-7) stimulation on renal dopamine uptake. Both anantin and cANP (4-23-amide) did not modify CNP effects on 3H-dopamine uptake by tubular cells. The Mas receptor antagonist, A-779, blocked the increase elicited by Ang-(1-7) on 3H-dopamine uptake. The stimulatory uptake induced by Ang-(1-7) was even more pronounced in the presence of losartan, suggesting an inhibitory effect of Ang-(1-7) on AT1 receptors on 3H-dopamine uptake. By increasing dopamine bioavailability in tubular cells, Ang-(1-7) enhances Na+, K+-ATPase activity inhibition, contributing to its natriuretic and diuretic effects.Considering the key role of renal dopamine in tubular sodium handling, we hypothesized that c-type natriuretic peptide (CNP) and Ang-(1-7) may regulate renal dopamine availability in tubular cells, contributing to Na Dopamin+-ATPase , 3.Diverse vasoactive peptides regulate blood pressure levels through modulation of renal function. In this way, members of the natriuretic peptide system, such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), c-type natriuretic peptide (CNP), and urodilatin (URO), regulate sodium excretion in the kidney for maintenance of the extracellular volume . CNP tha+-K+-ATPase [Ang-(1-7) is a biologically active heptapeptide that represents the main component of the depressor and protective arm of the renin angiotensin system . Besides+-ATPase .+, K+-ATPase activity [Renal dopamine may come from neuronal and extraneuronal sources. The extraneuronal source includes the uptake of dopamine from the blood and the tubular fluid, where the organic cation transporters (OCTs and OCTNs) play an important role . We haveactivity , 19. Thiactivity , 21. As activity , 23.+, K+-ATPase activity and to promote a greater natriuresis. In this sense, the aim of the present study was to investigate whether CNP and Ang-(1-7) could regulate dopamine uptake and Na+, K+-ATPase activity in renal tissue samples.Considering that CNP and Ang-(1-7) exert natriuretic and diuretic actions, we hypothesize that these peptides might regulate dopamine uptake by the tubular cells, controlling its bioavailability to bind D1 receptors. Therefore, both vasoactive peptides might collaborate with dopamine to enhance the inhibition of NaMale Sprague-Dawley rats  were housed at controlled temperature (23 ± 2°C) and exposed to a daily 12-hour light-dark cycle (lights on from 07:00 a.m. to 07:00 p.m.) with free access to tap water and standard rat chow  until the day of the experiment. Experiments were conducted in accordance with the international guiding principles and local regulations regarding the care and use of laboratory animals for biomedical research as well as the “International Ethical Guiding Principles for Biomedical Research on Animals” established by the CIOMS . The protocol was approved by the Institutional Committee for Care and Use of Laboratory Animals of the School of Pharmacy and Biochemistry of University of Buenos Aires . All surgery was performed under ethyl urethane anaesthesia and all efforts were made to minimize suffering.3H-dopamine and 28.0 Ci/mmol of specific activity ; CNP, hydrocortisone, nomifensine, anantin, des (Gln 18-Ser 19-Gly 20-Leu 21-Gly 22) atrial natriuretic peptide fragment 4-23-amide, Ang-(1-7), losartan, A-779, PD123319, carbidopa, imidazole, ATP (adenosine 5′ triphosphate), bovine seroalbumin fraction V of Cohn, and Folin reactive were from Sigma-Aldrich, Inc., Saint Louis, Missouri, USA. EcoLite, for liquid scintillation, was from ICN Pharmaceutical Inc., CA, USA.The following drugs and solutions were used in the experiments: 4·7H2O; 1.0 NaH2PO4; 2.4 CaCl2; 0.004 EDTA; 11.1 glucose; 0.11 ascorbic acid; 26.0 NaHCO3.Standard Krebs bicarbonate (SKB) solution of the following composition (mM) was used as incubation medium: 118 NaCl; 4.7 KCl; 1.2 MgSORats were anesthetized with 10% w/v ethyl urethane . Both kidneys were removed and washed with fresh SKB to remove the residual blood, and then slices of renal cortex and medulla were cut, minced, and weighed (approximately 40 mg).3H-dopamine uptake was measured as previously described by us [2 and 5% CO2 for 15 min; nomifensine (50 μM) was added in the preincubation medium to avoid neuronal dopamine uptake. Thereafter, tissues were transferred to fresh SKB and incubated, in similar conditions, with 0.625 μCi/mL of 3H-dopamine (22.32 nM), 50 μM nomifensine, and the different inhibitors for 15 min. After that time, CNP or Ang-(1-7) was added to the medium and the incubation continued for another 30 min. Control groups were incubated in the absence of the peptides. The following experiments were carried out in samples of renal cortex.ed by us . Briefly3H-dopamine uptake. The following groups were studied: control and incubated with 1, 10, and 100 pM and 1, 10, and 100 nM of CNP or Ang-(1-7).A concentration-response curve to CNP and Ang-(1-7) (1 pM to 100 nM) was performed to examine their effects on 3H-dopamine uptake at different times . The following groups were studied: control and incubated with 100 nM of CNP or 100 nM of Ang-(1-7).A time-course curve was carried out to study CNP and Ang-(1-7) effects on 3H-dopamine uptake in different areas of the kidney, 3H-dopamine uptake was determined in samples from renal cortex and medulla in the following groups: control and incubated with 100 nM of CNP or 100 nM of Ang-(1-7).To investigate CNP and Ang-(1-7) effects on 3H-dopamine uptake: (a) control and incubated with (b) 100 nM CNP; (c) 100 nM anantin (specific NPR-A receptor blocker); (d) 100 nM anantin plus 100 nM CNP; (e) 100 nM cANP (4-23-amide) (specific NPR-C receptor agonist); (f) 100 nM cANP (4-23-amide) plus 100 nM CNP.The following groups were studied in order to analyze if NPR-A or NPR-C receptors were involved in CNP effects on 3H-dopamine uptake, the following groups were studied: (a) control and incubated with (b) 100 nM Ang-(1-7); (c) 100 nM A-779 (specific Mas receptor blocker); (d) 100 nM A-779 plus 100 nM Ang-(1-7); (e) 100 nM PD123319 (specific AT2R blocker); (f) 100 nM PD123319 plus 100 nM Ang-(1-7); (g) 100 nM losartan (specific AT1R blocker); (h) 100 nM losartan plus 100 nM Ang-(1-7).To analyze if AT1R, AT2R, or Mas receptor was involved in Ang-(1-7) effects on 3H-dopamine uptake are expressed as d.p.m./g of fresh tissue.At the end of the incubation period, the tissues were washed with cold SKB solution, along 3 periods of 5 min each, and then homogenized with 2.5 mL of 10% trichloroacetic acid. The homogenates were centrifuged at 5000 rpm (1700 g) at 4°C for 30 min and tritium activity in the supernatants was determined by usual scintillation counting method. Results of +, K+-ATPase activity, the following experiments were performed in the presence of nomifensine  and carbidopa (to avoid dopamine synthesis). Carbidopa was administered in vivo  and in vitro . CNP as well as Ang-(1-7) effects were tested in the presence and in the absence of radio unlabeled dopamine and the nonneuronal dopamine uptake blocker hydrocortisone.To test whether the increase in renal dopamine produced by CNP or Ang-(1-7) could be associated with changes in NaμM carbidopa. The next groups were under 100 μM carbidopa: (c) 1 μM dopamine; (d) 100 nM CNP; (e) 100 nM Ang-(1-7); (f) 100 μM hydrocortisone; (g) 100 μM hydrocortisone plus 1 μM dopamine; (h) 100 nM CNP plus 1 μM dopamine; (i) 100 nM Ang-(1-7) plus 1 μM dopamine; (j) 100 nM CNP plus 1 μM dopamine plus 100 μM hydrocortisone; (k) 100 nM CNP plus 1 μM dopamine plus 100 μM hydrocortisone.The following groups were studied: (a) control and (b) incubated with 100 +, K+-ATPase activity was assayed in the supernatant as previously described [+, K+-ATPase activity, considering control values as 100%.Tissues were incubated for 30 min as described above and then homogenized (1 : 10 weight/volume) in 25 mM imidazole-1 mM EDTA-0.25 M sucrose solution and centrifuged at 5000 rpm (1700 g) at 4°C for 15 min. Naescribed . ATPase escribed , 25. Proescribed . Results t-test, one-way analysis of variance (ANOVA), and the Tukey test were performed when they corresponded. p values of 0.05 or less were considered statistically significant.All values are expressed as mean ± SEM. Data were processed using GraphPad in Stat Software . Student's3H-dopamine uptake in renal cortex. 10 nM and 100 nM Ang-(1-7) caused a significant increase in 3H-dopamine uptake. Then, we used the up threshold concentration of 100 nM Ang-(1-7) to continue the studies. On the other hand, CNP at any concentration used failed to alter 3H-dopamine uptake.3H-dopamine uptake between 5 and 45 min is displayed in 3H-dopamine uptake with 30-minute incubation period. On the contrary, CNP did not affect 3H-dopamine uptake at any time.The time course of 3H-dopamine uptake in renal cortex but lacked effects in renal medulla. CNP was not able to modify 3H-dopamine uptake of both renal cortex and medulla.3H-dopamine uptake in renal cortex [Considering that both ANP and URO increased l cortex , 19, andl cortex , we proc3H-dopamine uptake when NPR-A receptors were blocked by the selective NPR-A receptor blocker, 100 nM anantin. In 3H-dopamine uptake, suggesting that neither activation of NPR-A nor activation of NPR-B by CNP can affect this process. Moreover, the specific NPR-C agonist, 100 nM cANP (4-23-amide), neither altered dopamine uptake nor modified the effect of CNP on dopamine uptake.The next goal was to test whether CNP effects on dopamine uptake were mediated by activation of NPR-B receptors. Since CNP acts mainly through activation of NPR-B receptors, we wanted to discard that the lack of effects of CNP on dopamine uptake, shown in 3H-dopamine uptake in renal cortex, we proceeded to evaluate the participation of AT1R, AT2R, and Mas receptor by using different specific antagonist  on tagonist . The sel+, K+-ATPase activity in renal tubules, we assayed the effect of CNP and Ang-(1-7) added alone and together with dopamine on the enzyme activity. +, K+-ATPase activity increased by 56% compared with controls group. Under this condition, when dopamine or Ang-(1-7) was added alone, the enzyme activity diminished approximately by 40% and 25%, respectively, although when dopamine and Ang-(1-7) were added simultaneously, a significant further decrease in Na+, K+-ATPase was observed (approximately 60% of reduction compared with carbidopa group). On the other hand, the addition of hydrocortisone did not modify per se Na+, K+-ATPase activity as compared with carbidopa treated group, but the corticoid reversed the inhibitory action elicited by dopamine. Moreover, when dopamine and Ang-(1-7) were added simultaneously, hydrocortisone reversed partially the overinhibition elicited by dopamine and Ang-(1-7) on the enzyme activity. Finally, CNP by itself did not show any action on the Na+, K+-ATPase activity and did not modify the inhibitory action of dopamine on the enzyme activity.In order to determine whether the increased uptake of dopamine by CNP or Ang-(1-7) may modify Na+, K+-ATPase being one of the most important enzymes involved in sodium reabsorption in the kidney [The renal dopaminergic system is a local independent natriuretic system that contributes to preserving a normal balance of sodium and water, blood pressure levels, and renal redox steady state . It regue kidney . Howevere kidney –33. In te kidney . Therefoe kidney . Based o3H-dopamine uptake in renal cortex. The maximum effects observed at 30 minutes of incubation with a concentration of 100 nM Ang-(1-7) are in accordance with the results of previous experiments performed with other vasoactive peptides as ANP, URO, and Ang II [In this way, we have previously reported that ANP and URO stimulate dopamine uptake by the tubular cells in the kidney through NPR-A receptors coupled to guanylate cyclase and cGMP as second messenger , 21. As d Ang II , 19, 22.3H-dopamine uptake was only observed in the renal cortex, but not in medulla. In addition, the basal uptake of dopamine in medulla was even lower than that in renal cortex.Ang-(1-7) effects on dopamine uptake could be mediated by different mechanisms such as the increase of transporters, changes in cell membrane potential, and/or alteration in the carrier affinity. In this sense, renal OCTs have been described to transport dopamine from the bloodstream into the tubular cell , 36. TheWe also evaluated whether CNP, other member of the natriuretic peptide family, might share similar effect to that of ANP or URO on renal dopamine uptake. Our results show that CNP was not able to modify the dopamine uptake even at 45 minutes and also using high concentrations as 100 nM, in both renal cortex and medulla. This lack of effect of CNP on dopamine uptake may be related to different possibilities: CNP exerts its biological effects by selective activation of NPR-B receptors (that are expressed in a low proportion in the kidney), thereby increasing cGMP generation in target cells . In the +, K+-ATPase activity [Previous experimental evidences demonstrated that Ang-(1-7) decreased the release and increased the uptake of another catecholamine, norepinephrine, through activation of Mas receptors in the hypothalamus and brainstem of normotensive and SHR , 45. In activity . A dopamactivity , 51. Uriactivity . Altogetactivity –60. Claractivity . Ang-. This peptide binds to NPR-C receptors, which are devoid of kinase and guanylyl cyclase activities [The natriuretic peptides exert their biological actions through activation of NPR-A and/or NPR-B receptors ; meanwhitivities . The anaWhen comparing the potency of Ang-(1-7) with other vasoactive peptides that stimulate renal dopamine uptake , Ang- alone decreased significantly the sodium pump activity, and when it was added together with dopamine, they decreased further Na+, K+-ATPase activity, showing an additive effect on the sodium pump. To assess whether Ang-(1-7)-induced inhibition of Na+, K+-ATPase activity is related to Ang-(1-7) stimulation of extraneuronal uptake of dopamine, additional experiments were performed in the presence of hydrocortisone, a known inhibitor of extraneuronal uptake of amines. Hydrocortisone by itself reproduced similar levels of Na+, K+-ATPase activity as carbidopa but reversed the overinhibition of the enzyme induced by Ang-(1-7) plus dopamine. This finding confirms that Ang-(1-7) may stimulate dopamine uptake in the kidney, by regulating a typical hydrocortisone sensitive-extraneuronal uptake of amines.In order to elucidate whether CNP or Ang-(1-7) effects on dopamine uptake may modify renal sodium excretion, we determined the effects of these vasoactive peptides on the Na in vitro and also in vivo have suggested that Ang-(1-7) increases natriuresis and diuresis by regulating the activity of sodium transporters in the proximal tubule [+-K+-ATPase in isolated rat proximal tubules [−8 M), inhibits fluid absorption in isolated rat proximal straight tubules [+-K+-ATPase activity in the kidney. On the contrary, López Ordieres et al. showed that rat kidney Na+-K+-ATPase activity decreased roughly by 40–70% with 10−10 to 10−6 M Ang-(1-7) but increased by 22% with 10−12 M peptide concentration, thus indicating a biphasic effect [It must be pointed out that renal effects of Ang-(1-7) are complex to understand and may not always be opposite to those elicited by Ang II. In fact, Ang-(1-7) effects may be different and dependent on (a) the animal models used in the research; (b) the concentration reached by Ang-(1-7) at local or systemic levels; (c) the nephron segment where the study was performed; (d) the level of RAS activation; and (e) the sodium and water status in the kidney . Severall tubule . It has  tubules . Moreove tubules . These rc effect . In thisc effect , 70. Witc effect , 71. Thic effect .+-K+-ATPase activity. Moreover, when CNP was added together with dopamine, the natriuretic peptide showed an inhibitory effect on renal Na+, K+-ATPase as that observed when dopamine was used alone. These results demonstrate that CNP lacks regulating actions on Na+, K+-ATPase activity.Unlike Ang-(1-7), CNP, by itself, did not affect the NaThe natriuretic and diuretic effects of dopamine in the kidney are mediated by stimulation of D1 receptors, which are mainly intracellular and located in basal conditions . These rA complex interaction between AT1 and AT2 and Mas receptors should be taken into account to understand the sense of our results. Whether the increase in ACE2 and Ang-(1-7) might contribute to the beneficial effects of classical RAS inhibitors is still a matter of debate. In this sense, Ferrario et al. reported that treatment with losartan enhanced plasma and urinary concentrations of Ang-(1-7) and ACE2 activity in renal cortex, with no changes in the expression of ACE and in Mas and AT1 receptors . TherefoIn conclusion, the knowledge of the role that ACE2/Ang-(1-7)/Mas axis plays in the context of renal disease may be useful to prevent acute and chronic inflammation and fibrosis medications which target specifically the ACE2/Ang-(1-7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies , 82. Tak+, K+-ATPase activity. Dopamine and Ang-(1-7) may act in concert to inhibit tubular sodium reabsorption, as a consequence of increased dopamine uptake, resulting in overinhibition of Na+, K+-ATPase activity. In this way, Ang-(1-7) opposes one of the mechanisms of sodium transport regulation exerted by Ang II. Further studies must be performed in order to determine the intracellular events and signaling involved in Ang-(1-7)-dopamine relationship in the kidney. Our results demonstrate the existence of a close relationship between renal dopamine and vasoactive peptides, an interaction that might impact hydrosaline balance and whose impairment could be also involved in the pathogenesis of arterial hypertension.In summary, Ang-(1-7) stimulates dopamine uptake in renal cortex through activation of Mas receptor. Unlike other members of natriuretic peptides, CNP was not able to regulate renal dopamine uptake and Na