Massively parallel single-cell genomics of microbiomes in rice paddies

世界初のイネ根圏微生物叢の網羅的1細胞ゲノム解析に成功 --コメ生産現場が抱える問題のデータベース化に向けて--. 京都大学プレスリリース. 2022-11-09.Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and sustainable manner to ensure food security. Previous studies have attempted to understand the principles underlying the rhizosphere ecology and interactions between plants and PGPMs using ribosomal RNA sequencing, metagenomic sequencing, and genome-resolved metagenomics; however, these approaches do not provide comprehensive genomic information for individual species and do not facilitate detailed analyses of plant–microbe interactions. In the present study, we developed a pipeline to analyze the genomic diversity of the rice rhizosphere microbiome at single-cell resolution. We isolated microbial cells from paddy soil and determined their genomic sequences by using massively parallel whole-genome amplification in microfluidic-generated gel capsules. We successfully obtained 3, 237 single-amplified genomes in a single experiment, and these genomic sequences provided insights into microbial functions in the paddy ecosystem. Our approach offers a promising platform for gaining novel insights into the roles of microbes in the rice rhizomicrobiome and to develop microbial technologies for improved and sustainable rice production

Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

BACKGROUND: Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to determine the function of the hypothetical proteins. RESULTS: We examined 960 soluble proteins from Pyrococcus and selected 107 interactions based on luciferase reporter activity, which was then evaluated using a computational approach to assess the reliability of the interactions. We also analyzed the expression of the assay samples by western blot, and a few interactions by in vitro pull-down assays. We identified 11 hetero-interactions that we considered to be located at the same operon, as observed in Helicobacter pylori. We annotated and classified proteins in the selected interactions according to their orthologous proteins. Many enzyme proteins showed self-interactions, similar to those seen in other organisms. CONCLUSION: We found 13 unannotated proteins that interacted with annotated proteins; this information is useful for predicting the functions of the hypothetical Pyrococcus proteins from the annotations of their interacting partners. Among the heterogeneous interactions, proteins were more likely to interact with proteins within the same ortholog class than with proteins of different classes. The analysis described here can provide global insights into the biological features of the protein-protein interactions in P. horikoshii

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo− ) DNA polymerase

In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImON:NaNO and ImNO:NaON, using the Klenow fragment exo− [KF (exo−)]. We describe herein the successful results of (i) improved enzymatic recognition for ImNO:NaON base pairs and (ii) further primer extension reactions after the Im:Na base pairs by Deep Vent DNA polymerase exo− [Deep Vent (exo−)]. Since KF (exo−) did not catalyze primer extension reactions after the Im:Na base pair, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo−), seemed most promising for this purpose. In the ImON:NaNO base pair, incorporation of NaNOTP against ImON in the template was preferable to that of the natural dNTPs, while incorporation of dATP as well as dGTP competed with that of ImONTP when NaNO was placed in the template. Thus, the selectivity of base pair recognition by Deep Vent (exo−) was less than that by KF (exo−) in the case of the ImON:NaNO base pair. On the other hand, incorporation of NaONTP against ImNO in the template and that of ImNOTP against NaON were both quite selective. Thus, the selectivity of base pair recognition was improved by Deep Vent (exo−) in the ImNO:NaON base pair. Moreover, this enzyme catalyzed further primer extension reactions after the ImNO:NaON base pair to afford a faithful replicate, which was confirmed by MALDI-TOF mass spectrometry as well as the kinetics data for extension fidelity next to the ImNO:NaON base pair. The results presented in this paper revealed that the ImNO:NaON base pair might be a third base pair beyond the Watson–Crick base pairs

Ultrafast Control of Crystal Structure in a Topological Charge-Density-Wave Material

Optical control of crystal structures is a promising route to change physical properties including topological nature of a targeting material. Time-resolved X-ray diffraction measurements using the X-ray free-electron laser are performed to study the ultrafast lattice dynamics of VTe$_2$, which shows a unique charge-density-wave (CDW) ordering coupled to the topological surface states as a first-order phase transition. A significant oscillation of the CDW amplitude mode is observed at a superlattice reflection as well as Bragg reflections. The frequency of the oscillation is independent of the fluence of the pumping laser, which is prominent to the CDW ordering of the first-order phase transition. Furthermore, the timescale of the photoinduced 1$T^{\prime\prime}$ to 1$T$ phase transition is independent of the period of the CDW amplitude mode

Clinical Utility of Germline Genetic Testing in Japanese Men Undergoing Prostate Biopsy

Background: Multiple common variants and also rare variants in monogenic risk genes such as BRCA2 and HOXB13 have been reported to be associated with risk of prostate cancer (PCa); however, the clinical setting in which germline genetic testing could be used for PCa diagnosis remains obscure. Herein, we tested the clinical utility of a 16 common variant-based polygenic risk score (PRS) that has been developed previously for Japanese men and also evaluated the frequency of PCa-associated rare variants in a prospective cohort of Japanese men undergoing prostate biopsy. Methods: A total of 1336 patients undergoing first prostate biopsy were included. PRS was calculated based on the genotype of 16 common variants, and sequencing of 8 prostate cancer-associated genes was performed by multiplex polymerase chain reaction based target sequencing. PRS was combined with clinical factors in logistic regression models to assess whether addition of PRS improves the prediction of biopsy positivity. Results: The top PRS decile was associated with an odds ratio of 4.10 (95% confidence interval = 2.46 to 6.86) with reference to the patients at average risk, and the estimated lifetime absolute risk approached 20%. Among the patients with prostate specific antigen 2-10 ng/mL who had prebiopsy magnetic resonance imaging, high PRS had an equivalent impact on biopsy positivity as a positive magnetic resonance imaging finding. Rare variants were detected in 19 (2.37%) and 7 (1.31%) patients with positive and negative biopsies, respectively, with BRCA2 variants being the most prevalent. There was no association between PRS and high-risk rare variants. Conclusions: Germline genetic testing could be clinically useful in both pre- and post-PSA screening settings

Decline in Left Ventricular Ejection Fraction during Follow-up in Patients with Severe Aortic Stenosis

Objectives: The aim of this study was to investigate the prognostic impact of the decline in left ventricular ejection fraction (LVEF) at 1-year follow-up in patients with severe aortic stenosis (AS) managed conservatively. Background: No previous study has explored the association between LVEF decline during follow-up and clinical outcomes in patients with severe AS. Methods: Among 3, 815 patients with severe AS enrolled in the multicenter CURRENT AS (Contemporary Outcomes After Surgery and Medical Treatment in Patients With Severe Aortic Stenosis) registry in Japan, 839 conservatively managed patients who underwent echocardiography at 1-year follow-up were analyzed. The primary outcome measure was a composite of AS-related deaths and hospitalization for heart failure. Results: There were 91 patients (10.8%) with >10% declines in LVEF and 748 patients (89.2%) without declines. Left ventricular dimensions and the prevalence of valve regurgitation and atrial fibrillation or flutter significantly increased in the group with declines in LVEF. The cumulative 3-year incidence of the primary outcome measure was significantly higher in the group with declines in LVEF than in the group with no decline (39.5% vs. 26.5%; p 10% declines in LVEF at 1 year after diagnosis had worse AS-related clinical outcomes than those without declines in LVEF under conservative management. (Contemporary Outcomes After Surgery and Medical Treatment in Patients With Severe Aortic Stenosis Registry; UMIN000012140

Nonsense mutation in CFAP43 causes normal-pressure hydrocephalus with ciliary abnormalities

OBJECTIVE: To identify genes related to normal-pressure hydrocephalus (NPH) in one Japanese family with several members with NPH. METHODS:We performed whole-exome sequencing (WES) on a Japanese family with multiple individuals with NPH and identified a candidate gene.Then we generated knockout mouse using CRISPR/Cas9 to confirm the effect of the candidate gene on the pathogenesis of hydrocephalus.RESULTS: In WES, we identified a loss-of-function variant in CFAP43 that segregated with the disease. CFAP43 encoding cilia- and flagella-associated protein is preferentially expressed in the testis.Recent studies have revealed that mutations in this gene cause male infertility owing to morphologic abnormalities of sperm flagella. We knocked out mouse ortholog Cfap43 using CRISPR/Cas9 technology, resulting in Cfap43-deficient mice that exhibited a hydrocephalus phenotype with morphologic abnormality of motile cilia. CONCLUSION: Our results strongly suggest that CFAP43 is responsible for morphologic or movement abnormalities of cilia in the brain that result in NPH