Precipitant-Free Crystallization of Lysozyme and Glucose Isomerase by Drying

Protein crystallization is usually conducted by using precipitants, although the “salting-out” phenomenon is still unclear and complex. Moreover, the addition of precipitants sometimes results in irreversible disordered precipitation of protein molecules. Although precipitant-free lysozyme crystals obtained by centrifugal concentration showed significant changes in three-dimensional structure compared to the structure of salted-out crystals, it was rather difficult to mount crystals from a viscous dense liquid phase after centrifugal concentration, and the quality of the crystals often deteriorated during the mounting process. Here we present novel precipitant-free crystallization methods, which were effective for lysozyme and glucose isomerase. Tetragonal lysozyme crystals were successfully crystallized in a glass capillary simply by drying highly concentrated lysozyme solution in the presence of 0.01 M hydrochloric acid without using any precipitants. Glucose isomerase dissolved in ultra-pure water was also successfully crystallized in hanging drops by drying highly concentrated solution under low-humidity conditions. Oscillation images of the obtained crystals were safely collected without handling; they clearly indicated the crystals had a tetragonal form for lysozyme and an orthorhombic form for glucose isomerase, and their lattice parameters are similar to those of previously reported crystals obtained by salting-out methods

Warming and Sterilizing Towels by Microwave Irradiation

A steamed and sterilized towel was easily obtained using a home microwave oven. A small piece of gauze (7 × 7 cm) containing approximately 5 × 108 organisms in 1 mL of saline was rolled up in the moist towel which was wrapped in heat-resistant vinylidene polychloride film (wrap film). Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans in the towel were completely killed by microwave irradiation for 1 min. When three towels were irradiated simultaneously, it took 2 min for complete killing of the bacteria. In another experiment, the small gauze containing the bacterial suspension was set in a sterile plastic dish and completely dried using filtered air for a period of 3 h. After a 5-min irradiation in a microwave oven, the survival rate of S. aureus was 17%. These results indicate that a sterile steamed towel can be obtained by microwave irradiation of a moist towel wrapped in wrap film

Cellular response of Parachlorella kessleri to a solid surface culture environment

Attached culture allows high biomass productivity and is a promising biomass cultivating system because neither a huge facility area nor a large volume of culture medium are needed. This study investigates photosynthetic and transcriptomic behaviors in Parachlorella kessleri cells on a solid surface after their transfer from liquid culture to elucidate the physiological and gene-expression regulatory mechanisms that underlie their vigorous proliferation. The chlorophyll content shows a decrease at 12 h after the transfer; however, it has fully recovered at 24 h, suggesting temporary decreases in the amounts of light harvesting complexes. On PAM analysis, it is demonstrated that the effective quantum yield of PSII decreases at 0 h right after the transfer, followed by its recovery in the next 24 h. A similar changing pattern is observed for the photochemical quenching, with the PSII maximum quantum yield remaining at an almost unaltered level. Non-photochemical quenching was increased at both 0 h and 12 h after the transfer. These observations suggest that electron transfer downstream of PSII but not PSII itself is only temporarily damaged in solid-surface cells just after the transfer, with light energy in excess being dissipated as heat for PSII protection. It thus seems that the photosynthetic machinery acclimates to high-light and/or dehydration stresses through its temporal size-down and functional regulation that start right after the transfer. Meanwhile, transcriptomic analysis by RNA-Seq demonstrates temporary upregulation at 12 h after the transfer as to the expression levels of many genes for photosynthesis, amino acid synthesis, general stress response, and ribosomal subunit proteins. These findings suggest that cells transferred to a solid surface become stressed immediately after transfer but can recover their high photosynthetic activity through adaptation of photosynthetic machinery and metabolic flow as well as induction of general stress response mechanisms within 24 h

Kre6 (yeast 1,6-β-transglycosylase) homolog, PhTGS, is essential for β-glucan synthesis in the haptophyte Pleurochrysis haptonemofera

Haptophytes synthesize unique β-glucans containing more β-1,6-linkages than β-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-β-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed β-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in β-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where β-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into β-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage β-glucan synthesis specifically in light, probably by producing the β-1,6-branch

A hokistic understanfing of the human being : a report on student-volunteer activities at a pediatric ward

The authors of this paper planned and carried out a volunteer program for college students at a children\u27s ward in a university hospital, and subsequently analyzed students\u27 reports of their work in order to examine what the students had learned through the program. In the reports, the students initially expressed "anxiety," "fear," and "powerlessness" in a highly technological and medical environment, in which children and their family members were in crisis. However, through everyday discussion and reflection, they subsequently found new meaning in their work as volunteers, and writing about its significance of their work in terms of "keeping their eyes on the children" and "sitting close to the children." Through their experiences in such an environment, they seemed to confront their own vulnerability and reflect upon the deeper meaning of human nature and human relationships. These findings suggest that the students transformed their feelings of powerlessness in a crisis situation into a powerful driving-force for creating a new community.論文 (Article

BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains

The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target