Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

A significant outbreak of respiratory human adenovirus infections among children aged 3-6 years in Hokkaido, Japan, in 2023.

Human adenovirus (HAdV) infections present diverse clinical manifestations upon infecting individuals, with respiratory infections predominating in children. We surveyed pediatric hospitalizations due to respiratory HAdV infections across 18 hospitals in Hokkaido Prefecture, Japan, from July 2019 to March 2024, recording 473 admissions. While hospitalizations remained below five cases per week from July 2019 to September 2023, a notable surge occurred in late October 2023, with weekly admissions peaking at 15-20 cases from November to December. There were dramatic shifts in the age distribution of hospitalized patients: during 2019-2021, 1-year-old infants and children aged 3-6 years represented 51.4%-54.8% and 4.1%-13.3%, respectively; however, in 2023-2024, while 1-year-old infants represented 19.0%-20.1%, the proportion of children aged 3-6 years increased to 46.2%-50.0%. Understanding the emergence of significant outbreaks of respiratory HAdV infections and the substantial changes in the age distribution of hospitalized cases necessitates further investigation into the circulating types of HAdV in Hokkaido Prefecture and changes in children's neutralizing antibody titers against HAdV.

Intraspecific divergence in a coastal plant, Euphorbia jolkinii, at a major biogeographic boundary in East Asia.

PREMISE: Quaternary climatic fluctuations and long-distance seed dispersal across the sea are critical factors affecting the distribution of coastal plants, but the spatiotemporal nature of population expansion and distribution change of East Asian coastal plants during this period are rarely examined. To explore this process, we investigated the genome-wide phylogenetic patterns of Euphorbia jolkinii Boiss. (Euphorbiaceae), which grows widely on littoral areas of Japan, Korea, and Taiwan. METHODS: We used plastome sequences and genome-wide single nucleotide polymorphisms in samples across the species range to reveal phylogeographic patterns and spatiotemporal distributional changes. We conducted ecological niche modeling for the present and the last glacial maximum (LGM). RESULTS: Genetic differentiation was observed between the northern and southern populations of E. jolkinii, separated by the major biogeographic boundary, the Tokara Gap. These two groups of populations differentiated during the glacial period and subsequently intermingled in the intermorainic areas of the central Ryukyu Islands after the LGM. Ecological niche models suggested that the potential range of E. jolkinii was restricted to southern Kyushu; however, it was widespread in the southern Ryukyu Islands and Taiwan during the LGM. CONCLUSIONS: This study provides evidence of genetic differentiation among coastal plant populations separated by the prominent biogeographical boundary. Although coastal plants are typically expected to maintain population connectivity through sea-drifted seed dispersal, our findings suggest that genetic differences may arise because of a combination of limited gene flow and changes in climate during the glacial period.

Resurgence of human metapneumovirus infection and influenza after three seasons of inactivity in the post-COVID-19 era in Hokkaido, Japan, 2022-2023.

Following the coronavirus disease 2019 (COVID-19) outbreak in February 2020, incidences of various infectious diseases decreased notably in Hokkaido Prefecture, Japan. However, Japan began gradually easing COVID-19 infection control measures in 2022. Here, we conducted a survey of children hospitalized with human metapneumovirus (hMPV), influenza A and B, and respiratory syncytial virus infections in 18 hospitals across Hokkaido Prefecture, Japan, spanning from July 2019 to June 2023. From March 2020 to June 2022 (28 months), only 13 patients were hospitalized with hMPV, and two patients had influenza A. However, in October to November 2022, there was a re-emergence of hMPV infections, with a maximum of 27 hospitalizations per week. From July 2022 to June 2023 (12 months), the number of hMPV-related hospitalizations dramatically increased to 317 patients, with the majority aged 3-6 years (38.2%, [121/317]). Influenza A also showed an increase from December 2022, with a peak of 13 hospitalizations per week in March 2023, considerably fewer than the pre-COVID-19 outbreak in December 2019, when rates reached 45 hospitalizations per week. These findings suggest the possibility of observing more resurgences in infectious diseases in Japan after 2023 if infection control measures continue to be relaxed. Caution is needed in managing potential outbreaks.

Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B. × kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kozo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kozo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kozo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kozo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Neonatal suppurative parotitis: Case reports and literature review.

BACKGROUND: Neonatal suppurative parotitis is a rare disease, characterized mainly by unilateral parotid swelling with erythema and tenderness, and often purulent discharge from the Stensen's duct into the oral cavity. Only 44 cases were reported in the English literature between 1970 and 2013. METHODS: A MEDLINE search was conducted using the terms acute, neonatal, newborn, suppurative, bacterial, purulent, parotitis, parotid swelling, and parotid abscess, limited to the English-language literature starting from 2011. We reviewed all reported cases, together with two more managed cases in our hospital. We also describe the magnetic resonance imaging findings of the early stage of this disease. RESULTS: We identified 26 new cases since 2011. The total number of patients reviewed was 72, including our patients. The infection was unilateral in 83% of patients, and 67% of the affected patients were males. The serum amylase levels were generally not elevated despite marked parotid swelling. Of the causative agents of this disease, 65% were Staphylococcus aureus, of which 19% were methicillin-resistant S. aureus. As the rate of cesarean section was high in patients with this disease, it was considered a risk factor. The diffusion-weighted magnetic resonance images showed multiple punctate hyperintensity regions with reduced apparent diffusion coefficient, suggesting microabscess formation in the affected gland. CONCLUSIONS: Acute suppurative parotitis should be considered in cases of swelling and tenderness in the parotid gland during the neonatal period. Multiple punctate hyperintensities in the parotid gland on the diffusion-weighted images may indicate a retrograde bacterial infection from the Stensen's duct.

Sp7 Transgenic Mice with a Markedly Impaired Lacunocanalicular Network Induced Sost and Reduced Bone Mass by Unloading.

The relationship of lacunocanalicular network structure and mechanoresponse has not been well studied. The lacunocanalicular structures differed in the compression and tension sides, in the regions, and in genders in wild-type femoral cortical bone. The overexpression of Sp7 in osteoblasts resulted in thin and porous cortical bone with increased osteoclasts and apoptotic osteocytes, and the number of canaliculi was half of that in the wild-type mice, leading to a markedly impaired lacunocanalicular network. To investigate the response to unloading, we performed tail suspension. Unloading reduced trabecular and cortical bone in the Sp7 transgenic mice due to reduced bone formation. Sost-positive osteocytes increased by unloading on the compression side, but not on the tension side of cortical bone in the wild-type femurs. However, these differential responses were lost in the Sp7 transgenic femurs. Serum Sost increased in the Sp7 transgenic mice, but not in the wild-type mice. Unloading reduced the Col1a1 and Bglap/Bglap2 expression in the Sp7 transgenic mice but not the wild-type mice. Thus, Sp7 transgenic mice with the impaired lacunocanalicular network induced Sost expression by unloading but lost the differential regulation in the compression and tension sides, and the mice failed to restore bone formation during unloading, implicating the relationship of lacunocanalicular network structure and the regulation of bone formation in mechanoresponse.

Probing the Biogenesis of Polysaccharide Granules in Algal Cells at Sub-Organellar Resolution via Raman Microscopy with Stable Isotope Labeling.

Phototrophs assimilate CO2 into organic compounds that accumulate in storage organelles. Elucidation of the carbon dynamics of storage organelles could enhance the production efficiency of valuable compounds and facilitate the screening of strains with high photosynthetic activity. To comprehensively elucidate the carbon dynamics of these organelles, the intraorganellar distribution of the carbon atoms that accumulate at specific time periods should be probed. In this study, the biosynthesis of polysaccharides in storage organelles was spatiotemporally probed via stimulated Raman scattering (SRS) microscopy using a stable isotope (13C) as the tracking probe. Paramylon granules (a storage organelle of ß-1,3-glucan) accumulated in a unicellular photosynthetic alga, Euglena gracilis, were investigated as a model organelle. The carbon source of the culture medium was switched from NaH12CO3 to NaH13CO3 during the production of the paramylon granules; this resulted in the distribution of the 12C and 13C constituents in the granules, so that the biosynthetic process could be tracked. Taking advantage of high-resolution SRS imaging and label switching, the localization of the 12C and 13C constituents inside a single paramylon granule could be visualized in three dimensions, thus revealing the growth process of paramylon granules. We propose that this method can be used for comprehensive elucidation of the dynamic activities of storage organelles.

Preparation of cerium molybdates and their antiviral activity against bacteriophage Φ6 and SARS-CoV-2.

Two cerium molybdates (Ce2Mo3O12 and γ-Ce2Mo3O13) were prepared using either polymerizable complex method or hydrothermal process. The obtained powders were almost single-phase with different cerium valence. Both samples were found to have antiviral activity against bacteriophage Φ6. Especially, γ-Ce2Mo3O13 exhibited high antiviral activity against both bacteriophage Φ6 and SARS-CoV-2 coronavirus, which causes COVID-19. A synergetic effect of Ce and molybdate ion was inferred along with the specific surface area as key factors for antiviral activity.

Niche conservatism promotes speciation in cycads: the case of Dioon merolae (Zamiaceae) in Mexico.

Niche conservatism is the tendency of lineages to retain the same niche as their ancestors. It constrains biological groups and prevents ecological divergence. However, theory predicts that niche conservatism can hinder gene flow, strengthen drift and increase local adaptation: does it mean that it also can facilitate speciation? Why does this happen? We aim to answer these questions. We examined the variation of chloroplast DNA, genome-wide single nucleotide polymorphisms, morphological traits and environmental variables across the Dioon merolae cycad populations. We tested geographical structure, scenarios of demographic history, and niche conservatism between population groups. Lineage divergence is associated with the presence of a geographical barrier consisting of unsuitable habitats for cycads. There is a clear genetic and morphological distinction between the geographical groups, suggesting allopatric divergence. However, even in contrasting available environmental conditions, groups retain their ancestral niche, supporting niche conservatism. Niche conservatism is a process that can promote speciation. In D. merolae, lineage divergence occurred because unsuitable habitats represented a barrier against gene flow, incurring populations to experience isolated demographic histories and disparate environmental conditions. This study explains why cycads, despite their ancient lineage origin and biological stasis, have been able to diversify into modern ecosystems worldwide.

Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data.

BACKGROUND AND AIMS: The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. METHODS: We first compared the performance of phylogenetic analysis based on the plastid matK and trnL-F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. KEY RESULTS: Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0-84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. CONCLUSIONS: The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Isolating Single Euglena gracilis Cells by Glass Microfluidics for Raman Analysis of Paramylon Biogenesis.

Time-course analysis of single cells is important to characterize heterogeneous activities of individual cells such as the metabolic response to their environment. Single-cell isolation is an essential step prior to time-course analysis of individual cells by collecting, culturing, and identifying multiple single-cell targets. Although single-cell isolation has been performed by various methods previously, a glass microfluidic device with semiclosed microchannels dramatically improved this process with its simple operation and easy transfer for time-course analysis of identified single cells. This study demonstrates isolating single cells of the highly motile microalgae, Euglena gracilis, by semiclosed microchannels with liquid flow only. The isolated single cells were identified in isolating channels and continuously cultured to track, by Raman microscopy, for the formation of subcellular granules composed of polysaccharide paramylon, a unique metabolite of E. gracilis, generated through photosynthesis. Through low-temperature glass bonding, a thin glass interface was incorporated to the microfluidic device. Thus, the device could perform the direct measurements of cultured single cells at high magnification by Raman microscopy with low background noise. In this study, the first demonstration of sequential monitoring of paramylon biogenesis in a single identified E. gracilis cell is shown.

Patterns of population structure and complex haplotype sharing among field isolates of the green alga Chlamydomonas reinhardtii.

The nature of population structure in microbial eukaryotes has long been debated. Competing models have argued that microbial species are either ubiquitous, with high dispersal and low rates of speciation, or that for many species gene flow between populations is limited, resulting in evolutionary histories similar to those of macroorganisms. However, population genomic approaches have seldom been applied to this question. Here, we analyse whole-genome resequencing data for all 36 confirmed field isolates of the green alga Chlamydomonas reinhardtii. At a continental scale, we report evidence for putative allopatric divergence, between both North American and Japanese isolates, and two highly differentiated lineages within N. America. Conversely, at a local scale within the most densely sampled lineage, we find little evidence for either spatial or temporal structure. Taken together with evidence for ongoing admixture between the two N. American lineages, this lack of structure supports a role for substantial dispersal in C. reinhardtii and implies that between-lineage differentiation may be maintained by reproductive isolation and/or local adaptation. Our results therefore support a role for allopatric divergence in microbial eukaryotes, while also indicating that species may be ubiquitous at local scales. Despite the high genetic diversity observed within the most well-sampled lineage, we find that pairs of isolates share on average ~9% of their genomes in long haplotypes, even when isolates were sampled decades apart and from different locations. This proportion is several orders of magnitude higher than the Wright-Fisher expectation, raising many further questions concerning the evolutionary genetics of C. reinhardtii and microbial eukaryotes generally.

Optofluidic time-stretch quantitative phase microscopy.

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe.

Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D2 O) as a tracking probe. Specifically, we replaced H2 O in culture media with D2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D2 O as a tracking probe.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry.

Unique parallel radiations of high-mountainous species of the genus Sedum (Crassulaceae) on the continental island of Taiwan.

We explored the temporal and spatial diversification of the plant genus Sedum L. (Crassulaceae) in Taiwan based on molecular analysis of nrITS and cpDNA sequences from East Asian Sedum members. Our phylogenetic and ancestral area reconstruction analysis showed that Taiwanese Sedum comprised two lineages that independently migrated from Japan and Eastern China. Furthermore, the genetic distances among species in these two clades were smaller than those of other East Asian Sedum clades, and the Taiwanese members of each clade occupy extremely varied habitats with similar niches in high-mountain regions. These data indicate that species diversification occurred in parallel in the two Taiwanese Sedum lineages, and that these parallel radiations could have occurred within the small continental island of Taiwan. Moreover, the estimated time of divergence for Taiwanese Sedum indicates that the two radiations might have been correlated to the formation of mountains in Taiwan during the early Pleistocene. We suggest that these parallel radiations may be attributable to the geographical dynamics of Taiwan and specific biological features of Sedum that allow them to adapt to new ecological niches.

Effects of high CO2 on growth and metabolism of Arabidopsis seedlings during growth with a constantly limited supply of nitrogen.

Elevated CO2 has been reported to stimulate plant growth under nitrogen-sufficient conditions, but the effects of CO2 on growth in a constantly nitrogen-limited state, which is relevant to most natural habitats of plants, remain unclear. Here, we maintained Arabidopsis seedlings under such conditions by growing a mutant with reduced nitrate uptake activity on a medium containing nitrate as the sole nitrogen source. Under nitrogen-sufficient conditions (i.e. in the presence of ammonium), growth of shoots and roots of both the wild type (WT) and the mutant was increased approximately 2-fold by elevated CO2. Growth stimulation of shoots and roots by elevated CO2 was observed in the WT growing with nitrate as the sole nitrogen source, but in the mutant grown with nitrate, the high-CO2 conditions stimulated only the growth of roots. In the mutant, elevated CO2 caused well-known symptoms of nitrogen-starved plants, including decreased shoot/root ratio, reduced nitrate content and accumulation of anthocyanin, but also had an increased Chl content in the shoot, which was contradictory to the known effect of nitrogen depletion. A high-CO2-responsive change specific to the mutant was not observed in the levels of the major metabolites, although CO2 responses were observed in the WT and the mutant. These results indicated that elevated CO2 causes nitrogen limitation in the seedlings grown with a constantly limited supply of nitrogen, but the Chl content and the root biomass of the plant increase to enhance the activities of both photosynthesis and nitrogen uptake, while maintaining normal metabolism and response to high CO2.