Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

A methoxyflavanone from the medicinal herb Perilla frutescens down-modulates Th17 response and ameliorates collagen-induced arthritis.

IL-17-producing T-helper 17 (Th17) cells are involved in the pathogenesis of autoimmune disorders such as rheumatoid arthritis (RA). Here, we show that a methoxyflavanone from the Asian medicinal herb Perilla frutescens (termed Perilla-derived methoxyflavanone, PDMF) suppresses Th17 response and collagen-induced arthritis (CIA), an animal model of RA. We found that co-stimulation with PDMF suppressed Th17 cell differentiation and inhibited IL-17A secretion by differentiated Th17 cells. In vivo administration of PDMF to a CIA mouse model significantly ameliorated the development of RA-like joint symptoms, accompanied by decreased IL-17A production. Mechanistically, PDMF neither suppresses Th17-inducing IL-6 signaling nor reciprocally expands regulatory T (Treg) cells, but rather negatively regulates T-cell receptor (TCR) signaling-driven activation of Akt, which is another positive regulator of Th17 cell differentiation. These results suggest that PDMF is useful in preventing RA and the pro-inflammatory Th17 response.

Polyamine oxidase 7 is a terminal catabolism-type enzyme in Oryza sativa and is specifically expressed in anthers.

Polyamine oxidase (PAO), which requires FAD as a cofactor, functions in polyamine catabolism. Plant PAOs are classified into two groups based on their reaction modes. The terminal catabolism (TC) reaction always produces 1,3-diaminopropane (DAP), H2O2, and the respective aldehydes, while the back-conversion (BC) reaction produces spermidine (Spd) from tetraamines, spermine (Spm) and thermospermine (T-Spm) and/or putrescine from Spd, along with 3-aminopropanal and H2O2. The Oryza sativa genome contains seven PAO-encoded genes termed OsPAO1-OsPAO7. To date, we have characterized four OsPAO genes. The products of these genes, i.e. OsPAO1, OsPAO3, OsPAO4 and OsPAO5, catalyze BC-type reactions. Whereas OsPAO1 remains in the cytoplasm, the other three PAOs localize to peroxisomes. Here, we examined OsPAO7 and its gene product. OsPAO7 shows high identity to maize ZmPAO1, the best characterized plant PAO having TC-type activity. OsPAO7 seems to remain in a peripheral layer of the plant cell with the aid of its predicted signal peptide and transmembrane domain. Recombinant OsPAO7 prefers Spm and Spd as substrates, and it produces DAP from both substrates in a time-dependent manner, indicating that OsPAO7 is the first TC-type enzyme identified in O. sativa. The results clearly show that two types of PAOs co-exist in O. sativa. Furthermore, OsPAO7 is specifically expressed in anthers, with an expressional peak at the bicellular pollen stage. The physiological function of OsPAO7 in anthers is discussed.

Upper Respiratory Symptoms as Long COVID: Insight from a Multicenter Cohort Study.

Objective: This study aimed to investigate the clinical features of long COVID cases presenting with upper respiratory symptoms, a topic not yet fully elucidated. Study Design: Prospective cohort study. Setting: A multicenter study involving 26 medical facilities in Japan. Methods: Inclusion criteria were patients aged ≥18 years old with a confirmed COVID-19 diagnosis via severe acute respiratory syndrome coronavirus 2 polymerase chain reaction or antigen testing, who were hospitalized at the participating medical facilities. Analyzing clinical information and patient-reported outcomes from 1009 patients were analyzed. The outcome measured the degree of initial symptoms for taste or olfactory disorders and assessed the likelihood of these symptoms persisting as long COVID, as well as the impact on quality of life if the upper respiratory symptoms persisted as long COVID. Results: Patients with high albumin, low C-reactive protein, and low lactate dehydrogenase in laboratory tests tended to experience taste or olfactory disorders as part of long COVID. Those with severe initial symptoms had a higher risk of experiencing residual symptoms at 3 months, with an odds ratio of 2.933 (95% confidence interval [CI], 1.282-6.526) for taste disorders and 3.534 (95% CI, 1.382-9.009) for olfactory disorders. Presence of upper respiratory symptoms consistently resulted in lower quality of life scores. Conclusion: The findings from this cohort study suggest that severe taste or olfactory disorders as early COVID-19 symptoms correlate with an increased likelihood of persistent symptoms in those disorders as long COVID.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

High-throughput line-illumination Raman microscopy with multislit detection.

Raman microscopy is an emerging tool for molecular imaging and analysis of living samples. Use of Raman microscopy in life sciences is, however, still limited because of its slow measurement speed for spectral imaging and analysis. We developed a multiline-illumination Raman microscope to achieve ultrafast Raman spectral imaging. A spectrophotometer equipped with a periodic array of confocal slits detects Raman spectra from a sample irradiated by multiple line illuminations. A comb-like Raman hyperspectral image is formed on a two-dimensional detector in the spectrophotometer, and a hyperspectral Raman image is acquired by scanning the sample with multiline illumination array. By irradiating a sample with 21 simultaneous illumination lines, we achieved high-throughput Raman hyperspectral imaging of mouse brain tissue, acquiring 1108800 spectra in 11.4 min. We also measured mouse kidney and liver tissue as well as conducted label-free live-cell molecular imaging. The ultrafast Raman hyperspectral imaging enabled by the presented technique will expand the possible applications of Raman microscopy in biological and medical fields.

Morphometrics in three dimensional choroidal vessel models constructed from swept-source optical coherence tomography images.

We created three types of vessel models: vessel volume, surface, and line models from swept-source optical coherence tomography images and tested experimentally calculated three-dimensional (3D) biomarkers. The choroidal volume (CVolume), surface area (VSurface), and vessel length-associated index (VLI) were measured. The calculated 3D parameters were the mean choroidal thickness, choroidal vascularity index (CVI), vessel length density index (VLDI), vessel length to the stromal (VL-S) ratio, surface-to-volume ratio (S-V ratio), and vessel diameter index (VDI). Cluster analysis showed that the parameters were classified into two clusters: one was represented by the VVolume including the CVolume, VSurface, CVI, S-V ratio, VLI, VDI, and subfoveal choroidal thickness and the other by the VL-S ratio including the VLDI. Regarding the regional distribution, the VVolume, CVolume, VSurface, CVI, VLI, VL-S ratio, and VDI at the foveal center were higher than at the parafovea (P < 0.01). Although the VVolume decreased with age and axial length (AL) elongation, the association of the 3D parameters with age and AL elongation differed. The VLI, VLDI, VL-S ratio, and CVI decreased with age (P < 0.01) but not with AL elongation. The results suggested a structural difference in the choroidal vessel volume reduction between aging and AL elongation. The 3D parameters may provide additional information about the choroidal vasculature.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Comparative analysis of microRNA profiles of rice anthers between cool-sensitive and cool-tolerant cultivars under cool-temperature stress.

Plants subjected to abiotic stress can regulate gene expression post-transcriptionally by means of small RNAs such as microRNAs. Cool-temperature stress causes abnormal tapetum hypertrophy in rice anthers, leading to pollen sterility. As a first step toward understanding the molecular mechanisms of cool tolerance in developing anthers of rice, we report here a comprehensive comparative analysis of microRNAs between cool-sensitive Sasanishiki and cool-tolerant Hitomebore cultivars. High-throughput Illumina sequencing revealed 241 known and 46 novel microRNAs. Interestingly, 15 of these microRNAs accumulated differentially in the two cultivars at the uninucleate microspore stage under cool conditions. Inverse correlations between expression patterns of microRNAs and their target genes were confirmed by quantitative RT-PCR analysis, and cleavage sites of some of the target genes were determined by 5' RNA ligase-mediated RACE experiments. Thus, our data are useful resources to elucidate microRNA-mediated mechanism(s) of cool tolerance in rice anthers at the booting stage.