Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

<p>Abstract</p> <p>Background</p> <p>Various protein expression systems, such as <it>Escherichia coli </it>(<it>E. coli</it>), <it>Saccharomyces cerevisiae </it>(<it>S. cerevisiae</it>), <it>Pichia pastoris </it>(<it>P. pastoris</it>), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β₂adrenergic receptor, adenosine A_2areceptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, <it>P. pastoris </it>and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.</p> <p>Results</p> <p>The ideal conditions for the expression of CHRM2 in <it>P. pastoris </it>were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [³H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by <it>P. pastoris </it>was lower than that of Sf9 insect cells, CHRM2 yield in <it>P. pastoris </it>was 2-fold higher than in Sf9 insect cells because <it>P. pastoris </it>was cultured at high cell density. The dissociation constant (Kd) for QNB in <it>P. pastoris </it>was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between <it>P. pastoris </it>and Sf9 insect cells.</p> <p>Conclusion</p> <p>Compared to insect cells, <it>P. pastoris </it>is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, <it>P. pastoris</it>, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in <it>P. pastoris </it>can be applied to the structural and biochemical studies of GPCRs.</p

JASMINE: Near-infrared astrometry and time-series photometry science

The Japan Astrometry Satellite Mission for INfrared Exploration (JASMINE) is a planned M-class science space mission by the Institute of Space and Astronautical Science, the Japan Aerospace Exploration Agency. JASMINE has two main science goals. One is Galactic archaeology with a Galactic Center survey, which aims to reveal the Milky Way’s central core structure and formation history from Gaia-level (∼25 ${\mu}$as) astrometry in the near-infrared (NIR) Hw band (1.0–1.6 ${\mu}$m). The other is an exoplanet survey, which aims to discover transiting Earth-like exoplanets in the habitable zone from NIR time-series photometry of M dwarfs when the Galactic Center is not accessible. We introduce the mission, review many science objectives, and present the instrument concept. JASMINE will be the first dedicated NIR astrometry space mission and provide precise astrometric information on the stars in the Galactic Center, taking advantage of the significantly lower extinction in the NIR. The precise astrometry is obtained by taking many short-exposure images. Hence, the JASMINE Galactic Center survey data will be valuable for studies of exoplanet transits, asteroseismology, variable stars, and microlensing studies, including discovery of (intermediate-mass) black holes. We highlight a swath of such potential science, and also describe synergies with other missions

マメガキ(Diospyros lotus L.)における雌雄性判別分子マーカーの開発とカキ(D. kaki Thunb.)への適用

植物で初、雌雄異株性の性決定因子を柿において発見. 京都大学プレスリリース. 2014-10-31.Sexuality of crops affects both cultivation and breeding systems. Cultivated persimmon (Diospyros kaki Thunb) has a morphologically well-characterized polygamous or gyonodioecious sexual system. However, the genetic basis of sexuality in D. kaki has yet to be characterized. Here, we used dioecious D. lotus L., a diploid wild relative species to hexaploid or nonaploid D. kaki, as a model to clarify the genetic basis of sexuality in Diospyros and to develop molecular markers associated with the sexuality of individuals. Using 62 F1 offspring segregated into distinct male/female phenotypes, we found two amplified fragment-length polymorphism markers, DlSx-AF4 and DlSx-AF7, which cosegregated with maleness. This could suggest that the sexuality of D. lotus is controlled by a single gene/haploblock, and the male is dominant over the female. Thus, D. lotus’s sexuality can be described as the heterogametic male type, the XY-type, as reported for most other dioecious plant species. For unknown reasons, segregation of the phenotype of a sequence-characterized amplified region marker developed from DlSx-AF4 (DlSx-AF4S) and/or the male/female phenotype in two different crosses in D. lotus showed an apparent bias towards femaleness and better fitted 1:2 than 1:1, which is the theoretical segregation for a single genetic locus or haploblock in diploid D. lotus. DlSx-AF4S could distinguish D. kaki cultivars with female and male flowers from cultivars with only female flowers, strongly indicating that the same genetic system controls D. kaki’s sexuality and that DlSx-AF4S could be used as a genetic marker for sexuality in D. kaki breeding programs.雌雄性は作物の栽培そして育種の大きな制限要因となる．カキ（Diospyros kaki Thunb.）の雌雄性に関してこれまでに多くの形態学的な観察が行われ，カキは雌雄混株あるいは雌性両性異株とされている．しかしながら，カキにおける雌雄性の遺伝制御機構は明らかにされていない．本研究では，カキ（六倍体あるいは九倍体）の近縁野生種であり，雌雄異株であるマメガキ（D. lotus L.）（二倍体）を用い，交雑分離集団を作出し，雌雄分離の調査を行った．また同じ集団を用いて AFLP 解析によって雌雄性判別のための分子マーカーの開発を試みたところ，雄性と共分離を示す 2 種類の AFLP バンド（DlSx-AF4 および DlSx-AF7）が同定された．このため，マメガキの雌雄性は，他の多くの雌雄異株の植物種と同じく，単一の遺伝子座（ハプロブロック）によって支配される雄性異型接合型（XY 型）であることが明らかになった．しかし，DlSx-AF4 から作製した SCAR マーカー DlSx-AF4S および雌雄性形質の分離は，一遺伝子座支配における理論値である 1:1 から有意に雌性側に偏り，1:2 に近い分離を示した．DlSx-AF4S はカキにおいても雄花の着花性と相関を示し，カキの雌雄性もマメガキと同様の遺伝制御を受けていること，ならびにカキの育種における DlSx-AF4S を用いたマーカー選抜の可能性が示された

Epidemiology and Reporting Characteristics of Systematic Reviews in Orthopedic Journals: A Meta-Epidemiological Study

Systematic reviews (SRs) with complete reporting or rigorous methods can lead to less biased recommendations and decisions. A comprehensive analysis of the epidemiological and reporting characteristics of SRs in orthopedics is lacking. We evaluated 360 SRs, including 165 and 195 published in orthopedic journals in 2012 and 2022. According to the established reporting guidelines, we examined these SRs for key epidemiological characteristics, including focus areas, type of meta-analysis (MA), and reporting characteristics. Most SRs (71%) were therapy-related, with a significant proportion originating from authors in the USA, UK, and China. Pairwise MA was performed on half of the SRs. The proportion of protocol registrations improved by 2022 but remained low (33%). Despite a formal declaration of adherence to the reporting guidelines (68%), they were often not used and reported enough. Only 10% of the studies used full search strategies, including trial registries. Publication bias assessments, subgroup analyses, and sensitivity analyses were not even planned. The risk of bias assessment improved in 2022; however, the certainty of the evidence remained largely unassessed (8%). The use and reporting of standard methods in orthopedic SRs have remained suboptimal. Thus, authors, peer reviewers, journal editors, and readers should criticize the results more