Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4 + T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.

Anode-supported thin electrolyte cells are studied by electrochemical impedance spectroscopy ͑EIS͒. The aim is to describe how the losses of this type of cells are distributed at low current density ͑around open-circuit voltage͒ as a function of temperature. An equivalent circuit consisting of an inductance, a serial resistance ͑R s ͒, and five arcs to describe the polarization resistance is suggested. This equivalent circuit is based on previous studies of single electrodes in three-electrode and two-electrode symmetric cell setups. The equivalent circuit components have been assigned to the electrode processes, and the assignments were verified by extensive full cell studies in which the partial pressure of reactant gases on both the electrodes as well as temperature was systematically varied with the aim to identify frequency regions which are dominated by an electrode specific process. Furthermore, the model is applied on a good performing cell with area specific resistance ͑ASR͒ = 0.15 ⍀ cm 2 at 850°C and a poor performing cell with ASR = 0.29 ⍀ cm 2 at the same temperature. Both cells were fabricated using nominally the same procedure. The EIS analysis indicated that the difference in performance originates from microstructural differences on the cathode. This is further supported by the observation of large differences in the cathode microstructure by scanning electron microscope.

Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4 + T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.

The degradation behavior of anode supported solid oxide fuel cells ͑SOFCs͒ was investigated as a function of operating temperature and current density. Degradation rates were defined and shown to be mainly dependent on the cell polarization. The combination of a detailed evaluation of electrochemical properties by impedance spectroscopy, in particular, and post-test microscopy revealed that cathode degradation was the dominant contribution to degradation at higher current densities and lower temperatures. The anode was found to contribute more to degradation at higher temperatures. Generally, the degradation rates obtained were lower at higher operating temperatures, even at higher current densities. A degradation rate as low as 2%/1000 h was observed at 1.7 A/cm 2 and 950°C over an operating period of 1500 h.

The achievement of structural color has shown advantages in large-gamut, high-saturation, high-brightness, and high-resolution. While a large number of plasmonic/dielectric nanostructures have been developed for structural color, the previous approaches fail to match all the above criterion simultaneously. Herein we utilize the Si metasurface to demonstrate an all-in-one solution for structural color. Due to the intrinsic material loss, the conventional Si metasurfaces only have a broadband reflection and a small gamut of 78% of sRGB. Once they are combined with a refractive index matching layer, the reflection bandwidth and the background reflection are both reduced, improving the brightness and the color purity significantly. Consequently, the experimentally demonstrated gamut has been increased to around 181.8% of sRGB, 135.6% of Adobe RGB, and 97.2% of Rec.2020. Meanwhile, high refractive index of silicon preserves the distinct color in a pixel with 2 × 2 array of nanodisks, giving a diffraction-limit resolution.

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INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders. Synthesis, cyclization, and characterization of synV . ( A ) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. ( B ) CRISPR/Cas9 strategy for multiplex repair. ( C ) Colonies of wtV, synV, and ring_synV strains.

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