Rapid preparation of mutated influenza hemagglutinins for influenza virus pandemic prevention

Influenza viruses have periodically caused pandemic due to frequent mutation of viral proteins. Influenza viruses have two major membrane glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin plays a crucial role in viral entry, while NA is involved in the process of a viral escape. In terms of developing antiviral drugs, HA is a more important target than NA in the prevention of pandemic, since HA is likely to change the host specificity of a virus by acquiring mutations, thereby to increase the risk of pandemic. To characterize mutated HA functions, current approaches require immobilization of purified HA on plastic wells and carriers. These troublesome methods make it difficult to respond to emerging mutations. In order to address this problem, a yeast cell surface engineering approach was investigated. Using this technology, human HAs derived from various H1N1 subtypes were successfully and rapidly displayed on the yeast cell surface. The yeast-displayed HAs exhibited similar abilities to native influenza virus HAs. Using this system, human HAs with 190E and 225G mutations were shown to exhibit altered recognition specificities from human to avian erythrocytes. This system furthermore allowed direct measurement of HA binding abilities without protein purification and immobilization. Coupled with the ease of genetic manipulation, this system allows the simple and comprehensive construction of mutant protein libraries on yeast cell surface, thereby contributing to influenza virus pandemic prevention

Serum amyloid A triggers the mosodium urate -mediated mature interleukin-1β production from human synovial fibroblasts

Background: Monosodium urate (MSU) has been shown to promote inflammasome activation and interleukin-1β (IL-1β) secretion in monocyte/macrophages, but the cellular pathway and nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in synovial tissues, remain elusive. In this study, we investigated the effects of MSU on synovial fibroblasts to elucidate the process of MSU-mediated synovial inflammation.Methods: Human synovial fibroblasts were stimulated with MSU in the presence or absence of serum amyloid A (SAA). The cellular supernatants were analyzed by immunoblotting using anti-IL-1β or anti-caspase-1 antibodies. IL-1β or NLRP3 mRNA expressions were analyzed by real-time PCR or reverse transcription-PCR (RT-PCR) method.Results: Neither SAA nor MSU stimulation resulted in IL-1β or interleukin-1α (IL-1α) secretions and pro-IL-1β processing in synovial fibroblasts. However, in SAA-primed synovial fibroblasts, MSU stimulation resulted in the activation of caspase-1 and production of active IL-1β and IL-1α. The effect of SAA on IL-1β induction was impaired in cells by silencing NLRP3 using siRNA or treating with caspase-1 inhibitor. In addition, SAA induced the secretion of cathepsin B and NLRP3 mRNA expression in synovial fibroblasts.Conclusions: Our data demonstrate that exposure of human synovial fibroblasts to SAA promotes MSU-mediated caspase-1 activation and IL-1β secretion in the absence of microbial stimulation. These findings provide insight into the molecular processes underlying the synovial inflammatory condition of gout

Prevention of hypoglycemia by intermittent-scanning continuous glucose monitoring device combined with structured education in patients with type 1 diabetes mellitus : A randomized, crossover trial

Aims: We conducted a randomized, crossover trial to compare intermittent-scanning continuous glucose monitoring (isCGM) device with structured education (Intervention) to self-monitoring of blood glucose (SMBG) (Control) in the reduction of time below range. Methods: This crossover trial involved 104 adults with type 1 diabetes mellitus (T1DM) using multiple daily injections. Participants were randomly allocated to either sequence Intervention/Control or sequence Control/Intervention. During the Intervention period which lasted 84 days, participants used the first-generation FreeStyle Libre (Abbott Diabetes Care, Alameda, CA, USA) and received structured education on how to prevent hypoglycemia based on the trend arrow and by frequent sensor scanning (≥10 times a day). Confirmatory SMBG was conducted before dosing insulin. The Control period lasted 84 days. The primary endpoint was the decrease in the time below range (TBR; <70 mg/dL). Results: The time below range was significantly reduced in the Intervention arm compared to the Control arm (2.42 ± 1.68 h/day [10.1 %±7.0 %] vs 3.10 ± 2.28 h/day [12.9 %±9.5 %], P = 0.012). The ratio of high-risk participants with low blood glucose index >5 was significantly reduced (8.6 % vs 23.7 %, P < 0.001). Conclusions: The use of isCGM combined with structured education significantly reduced the time below range in patients with T1DM

酵母Saccharomyces cerevisiaeの熱適応戦略の解明と熱耐性の再構築

京都大学0048新制・課程博士博士(農学)甲第20439号農博第2224号新制||農||1049(附属図書館)学位論文||H29||N5060(農学部図書室)京都大学大学院農学研究科応用生命科学専攻(主査)教授 植田 充美, 教授 間藤 徹, 教授 喜多 恵子学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Generation of a functionally distinct Rhizopus oryzae lipase through protein folding memory

アンフィンセン説を覆す酵素タンパク質リパーゼの創製に成功. 京都大学プレスリリース. 2015-05-18.Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROL[imp]) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROL[imp] that exhibited different substrate specificities compared with mROL[WT] (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROL[imp] showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROL[imp] was more stable than mROL[WT]. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity

Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains

酵母の熱適応進化の大規模ゲノム解析によって熱耐性因子の同定に成功 －バイオエタノール生産性の飛躍的な向上に貢献－. 京都大学プレスリリース. 2016-03-22.Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction

Ketosis-prone Type 2 Diabetes(KPD)におけるインスリンペプチド特異的インターフェロンγ関連免疫応答の臨床的意義

令和4年

Sequence alignment of <i>Rhizopus oryzae</i> lipase (ROL) and ROL-related lipases.

<p>The full-length primary sequences of ROL, <i>R</i>. <i>niveus</i> lipase (RNL), <i>R</i>. <i>stolonifer</i> lipase (RSL), and <i>R</i>. <i>chinensis</i> lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, <i>R</i>. <i>oryzae</i>, the propeptide is also cleaved between the Ala97 and Ser98 residues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref021" target="_blank">21</a>]; however in <i>P</i>. <i>pastoris</i> and <i>S</i>. <i>cerevisiae</i>, the secondary cleavage has not been observed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref022" target="_blank">22</a>]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.</p