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.

Background: Cardiovascular disease (CVD) and colon cancer incidence are known to be closely related to dietary factors. This article evaluated effects of krill oil (KO) on serum lipids of hyperlipidemia rats and human colon cancer cells (SW480). Serum lipids of rats fed with high fat diet (HFD) and different doses of KO were measured by automatic analyzer. Effect of KO on viability of cells was determined by methyl thiazolyl tetrazolium (MTT) assay.

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Chimonanthus nitens Oliv. (CN) is a species in the family Calycanthaceae. Its leaf is widely used to make traditional herbal tea in southern China and has a wide range of therapeutic effects. The profile of the ethanol extracts from CN leaves was identified by UPLC-QTOF-MS/MS. Forty seven compounds were determined including organic acids, phenolic acids and derivatives, flavonoids, coumarins, fatty acids and other compounds. The effect of the CN extracts on the inflammatory damage in zebrafish and in RAW 264.7 cells was investigated. The extracts demonstrated a strong ability to inhibit the recruitment of neutrophils in LPS-stimulated zebrafish, but macrophage migration was not significantly affected. Pro-inflammatory cytokines (i.e., TNF-α, IL-6 and IL-1β) were also determined by q-PCR. The extracts strongly reduced mRNA expression of TNF-α, IL-6 but not IL-1β in zebrafish model, while significantly inhibited the production of the factors in the RAW 264.7 cells. Therefore, our results suggest that the ethanol extracts of CN leaves may serve as a source of nutraceutical compounds with anti-inflammatory properties.

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Owing to the increasing demand for tribological brakes for space applications, the development of novel materials and advanced technologies is necessary. This paper presents the design, characterization, and realization of powder metallurgy processed metal-matrix friction materials intended for the above-mentioned tribological brakes. Selecting appropriate ingredients, which provides an effective way to tailor the properties of the friction material, is evolving as a strategy to meet the design requirements. The tribological behaviors of the friction material are experimentally investigated under different conditions, and special attention is focused on the vacuum tribology. Examinations and analyses of the friction surface and subsurface corroborate the wear mechanism. In addition, the erosion resistances of the friction material are evaluated by exposure tests of ultraviolet irradiation and atomic oxygen. Finally, present and potential space applications of the friction material are also introduced based on experimental studies.

Fatty aldehydes and alcohols are valuable precursors used in the industrial manufacturing of a myriad of specialty products. Herein, we demonstrate the de novo production of odd chain-length fatty aldehydes and fatty alcohols in Saccharomyces cerevisiae by expressing a novel biosynthetic pathway involving cytosolic thioesterase, rice α-dioxygenase and endogenous aldehyde reductases. We attained production titers of ∼20 mg/l fatty aldehydes and ∼20 mg/l fatty alcohols in shake flask cultures after 48 and 60 h respectively without extensive fine-tuning of metabolic fluxes. In contrast to prior studies which relied on bi-functional fatty acyl-CoA reductase to produce even chain-length fatty alcohols, our biosynthetic route exploits α-oxidation reaction to produce odd chain-length fatty aldehyde intermediates without using NAD(P)H cofactor, thereby conserving cellular resource during the overall synthesis of odd chain-length fatty alcohols. The biosynthetic pathway presented in this study has the potential to enable sustainable and efficient synthesis of fatty acid-derived chemicals from processed biomass.