Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′-phosphotransferase-Ia [APH(7′′)-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7′′)-Ia and APH(4)-Ia, the crystal structure of APH(7′′)-Ia complexed with HygB is reported. The overall structure of APH(7′′)-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes

Effect of diamagnetic contribution of water on harmonics distribution in a dilute solution of iron oxide nanoparticles measured using high-Tc SQUID magnetometer

The magnetization curve of iron oxide nanoparticles in low-concentration solutions was investigated by a highly sensitive high-Tc superconducting quantum interference device (SQUID) magnetometer. The diamagnetic contribution of water that was used as the carrier liquid was observed in the measured magnetization curves in the high magnetic field region over 100 mT. The effect of the diamagnetic contribution of water on the generation of harmonics during the application of AC and DC magnetic fields was simulated on the basis of measured magnetization curves. Although the diamagnetic effect depends on concentration, a linear relation was observed between the detected harmonics and concentration in the simulated and measured results. The simulation results suggested that improvement could be expected in harmonics generation because of the diamagnetic effect when the iron concentration was lower than 72 μg/ml. The use of second harmonics with an appropriate bias of the DC magnetic field could be utilized for realization of a fast and highly sensitive detection of magnetic nanoparticles in a low-concentration solution

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

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

17 beta-Hydroxysteroid dehydrogenase type 12 is responsible for maturation-inducing steroid synthesis during oocyte maturation in Nile tilapia

17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of 17OHP to DHP by ovarian follicles incubated with SPE and HCG peaked at 16 h, subsequent with increased follicular hsd17b12 mRNA levels, which were significantly higher than those in control incubations. However, the levels of CR/20bhsd mRNA remained low and did not differ among time points. The present study strongly suggests that 17 beta-HSD12, and not CR/20 beta-HSD, is the 20 beta-FISD responsible for DHP production by ovarian follicles during oocyte maturation in Nile tilapia

Nurses' and students' perception of risk from medical practices

Objectives: To determine the risk perceptions of a series of medical practices in non-expert (undergraduates) and expert (nurses) samples. Methods: Four hundred and forty-seven nurses and 246 undergraduate students participated in this study. They all answered questionnaires about the risk dimensions and acceptance for medical practices. Results: An exploratory factor analysis on participants' answers to various dimensions of risk yielded a two-factor structure for risk perception in both samples: for nurses, the factors were “Unknown” and “Dread,” while for students, they were “Dread” and “Lack of Independence.” For both nurses and students, the factor scores of Dread negatively related to individual risk acceptance of medical practices. Furthermore, nurses tended to be more accepting of practices that they knew well (i.e., low Unknown scale scores). For students, the subscale scores of the Lack of Independence factor negatively related to individual risk acceptance only for health examination practices. Nurses conceived risks more correctly and concretely compared to students. This was especially pronounced for practices related to medication use. Conclusions: Although both nurses and students conceived various risk contents from medical practices, their conceptions still differed. Knowledge of these differences in the structure of risk perception and conceived risk contents of various medical practices between nurses and students could be utilized to improve risk communication in clinical practice

Regulation of 17 alpha-Hydroxyprogesterone Production during Induced Oocyte Maturation and Ovulation in Amur Sturgeon (Acipenser schrenckii)

In several teleosts, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) has been identified as a maturation-inducing steroid. DHP is synthesized from 17 alpha-hydroxyprogesterone (17OHP) by 17 beta-hydroxysteroid dehydrogenase type 12-like (hsd17b12L). Along with 3 beta-hydroxysteroid dehydrogenase/& UDelta;5-4 isomerase (3 beta-HSD), 17 alpha-hydroxylase and C17-20 lyase are associated with 17OHP production. This study aimed to determine the roles of Amur sturgeon hsd3b, P450c17-I (cyp17a1), and P450c17-II (cyp17a2) in 17OHP production and to examine their enzyme activity and mRNA expression pattern during oocyte maturation. In the sturgeons used in this study, hsd3b encoded 3 beta-HSD, cyp17a1 catalyzed 17 alpha-hydroxylase production with C17-20 lyase activity, and cyp17a2 processed 17 alpha-hydroxylase activity alone. In the ovarian follicles of individuals that underwent induced ovulation, hsd3b mRNA levels increased rapidly, cyp17a1 expression was downregulated, and cyp17a2 expression was upregulated during oocyte maturation. Finally, an in vitro study revealed that salmon pituitary extract (SPE) stimulation rapidly induced hsd3b expression, whereas cyp17a1 expression was downregulated. In vitro, cyp17a2 expression did not rapidly increase with SPE stimulation. This rapid upregulation of hsd3b during oocyte maturation was first observed in teleosts. It was suggested that hsd17b12L expression is upregulated after 17OHP production, which is regulated by hsd3b, cyp17a1, and cyp17a2, resulting in DHP production

Changes in Ovulation-Related Gene Expression during Induced Ovulation in the Amur Sturgeon (Acipenser schrenckii) Ovarian Follicles

The luteinizing hormone (LH) and maturation-inducing steroids (MIS), such as 17α,20β-dihydroxy-4-pregnen-3-one, regulate the final oocyte maturation in teleosts. Oocyte maturational competence (OMC) and ovulatory competence measure the sensitivity to MIS for oocyte maturation and ovulation, respectively. However, the molecular mechanisms underlying the acquisition of ovulatory competence remain unknown. Sturgeons are an excellent research model for investigating these mechanisms. We examined the seasonal profiles of OMC and ovulatory competence in vitro and the expression of 17 ovulation-related gene candidates using quantitative PCR in Amur sturgeon ovarian follicles. The ovulatory competence was induced by the LH-releasing hormone analog (LHRHa) priming injection after acquiring the OMC, which was spontaneously induced in spring or autumn. Seven genes, including the tissue-type plasminogen activator (plat), were enhanced following the LHRHa priming injection in ovarian follicles sampled from anovulated and ovulated fish. The activin receptor type 1 (acvr1) and prostaglandin G/H synthase 2 (ptgs2) were only upregulated in ovulated fish. Our results suggest that plat/plasmin and prostaglandin (PG)/PG receptor systems are essential for sturgeon ovulation, similar to other vertebrates. Notably, successful ovulation depends on a sufficient PG synthesis, and mediators activating the PG/PG receptor system are essential for acquiring the ovulatory competence. We provide the first report of ovulation-related gene alterations in the ovarian follicles of Amur sturgeons

Development of new small-angle neutron scattering geometry with ring-shaped collimated beam for compact neutron source

Small-angle neutron scattering (SANS) is an important tool to investigate material properties in nanometer to micrometer scale. The opportunity to conduct SANS experiments is, however, limited because of the low number of available SANS beam lines. Compact neutron sources are expected to play a significant role to increase neutron scattering facilities including SANS beam lines. The problem is that the flux of compact neutron sources can be very low, which makes it difficult to measure scattered neutrons from a sample. A SANS geometry with ring-shaped collimated beam (r-SANS) is developed to conduct SANS experiments at very low flux neutron sources. By using ring-shaped collimated neutrons to hit a large sample, the scattered neutron flux becomes high on the ring center line because the scattered neutrons with each scattered angle overlaps on each point of the ring center line. By setting a 3He point detector on the center line and shielding the surrounding of the small detection area well, high signal to noise ratio experiments are possible. In this paper, we show the concept of this new geometry and a preliminary experimental result of a glassy carbon sample taken with the r-SANS geometry constructed at Kyoto University proton Accelerator Neutron Source (KUANS)