Individual Atg8 paralogs and a bacterial metabolite sequentially promote hierarchical CASM-xenophagy induction and transition.

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.

Analysis of microbial dynamics in the soybean root-associated environments from community to single-cell levels.

Plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere, are notably different from non-root-associated soil environments. However, the microbial dynamics in these spatially divided compartments remain unexplored. In this study, we propose a combinational analysis of single-cell genomics with 16S rRNA gene sequencing. This method enabled us to understand the entire soil microbiome and individual root-associated microorganisms. We applied this method to soybean microbiomes and revealed that their composition was different between the rhizoplane and rhizosphere in the early growth stages, but became more similar as growth progressed. In addition, a total of 610 medium- to high-quality single-amplified genomes (SAGs) were acquired, including plant growth-promoting rhizobacteria (PGPR) candidates while genomes with high GC content tended to be missed by SAGs. The whole-genome analyses of the SAGs suggested that rhizoplane-enriched Flavobacterium solubilizes organophosphate actively and Bacillus colonizes roots more efficiently. Single-cell genomics, together with 16S rRNA gene sequencing, enabled us to connect microbial taxonomy and function, and assess microorganisms at a strain resolution even in the complex soil microbiome.

Comparative single-cell genomics of Atribacterota JS1 in the Japan Trench hadal sedimentary biosphere.

Deep-sea and subseafloor sedimentary environments host heterotrophic microbial communities that contribute to Earth's carbon cycling. However, the potential metabolic functions of individual microorganisms and their biogeographical distributions in hadal ocean sediments remain largely unexplored. In this study, we conducted single-cell genome sequencing on sediment samples collected from six sites (7,445-8,023 m water depth) along an approximately 500 km transect of the Japan Trench during the International Ocean Discovery Program Expedition 386. A total of 1,886 single-cell amplified genomes (SAGs) were obtained, offering comprehensive genetic insights into sedimentary microbial communities in surface sediments (<1 m depth) above the sulfate-methane transition zone along the Japan Trench. Our genome data set included 269 SAGs from Atribacterota JS1, the predominant bacterial clade in these hadal environments. Phylogenetic analysis classified SAGs into nine distinct phylotypes, whereas metagenome-assembled genomes were categorized into only two phylotypes, advancing JS1 diversity coverage through a single cell-based approach. Comparative genomic analysis of JS1 lineages from different habitats revealed frequent detection of genes related to organic carbon utilization, such as extracellular enzymes like clostripain and α-amylase, and ABC transporters of oligopeptide from Japan Trench members. Furthermore, specific JS1 phylotypes exhibited a strong correlation with in situ methane concentrations and contained genes involved in glycine betaine metabolism. These findings suggest that the phylogenomically diverse and novel Atribacterota JS1 is widely distributed in Japan Trench sediment, playing crucial roles in carbon cycling within the hadal sedimentary biosphere.IMPORTANCEThe Japan Trench represents tectonically active hadal environments associated with Pacific plate subduction beneath the northeastern Japan arc. This study, for the first time, documented a large-scale single-cell and metagenomic survey along an approximately 500 km transect of the Japan Trench, obtaining high-quality genomic information on hadal sedimentary microbial communities. Single-cell genomics revealed the predominance of diverse JS1 lineages not recoverable through conventional metagenomic binning. Their metabolic potential includes genes related to the degradation of organic matter, which contributes to methanogenesis in the deeper layers. Our findings enhance understanding of sedimentary microbial communities at water depths exceeding 7,000 m and provide new insights into the ecological role of biogeochemical carbon cycling in the hadal sedimentary biosphere.

Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids.

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high-risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR-GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR-GFP vectors were transduced into patient-derived squamocolumnar junction organoids, and the presence of GFP-positive cells was evaluated. Single-cell RNA sequencing of GFP-positive and GFP-negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP-positive and GFP-negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP-positive cells, whereas keratinization and mitophagy/autophagy-related pathways were upregulated in GFP-negative cells. siRNA-mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV-related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18-infected cells downregulated stem cell-related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18-infected cervical lesions.

Glutamate-Sensing Genes Are Conserved among Populations Compared to Glutamate Metabolism Genes.

INTRODUCTION: Glutamate is a representative taste molecule with an umami flavor and is a major nutrient found abundantly in nature. Furthermore, it plays a significant role in the human body as a key metabolic intermediate and neurotransmitter. Therefore, the divergence of glutamate functions among populations during their evolution is of particular interest with a hypothesis that the genetic variation can lead to understanding divergence in taste perception. To elucidate variation in glutamate applications and to deepen our understanding of taste perception, we examined the nucleotide diversity of genes associated with glutamate sensing and metabolism among human populations. METHODS: We first established 67 genes related to glutamate sensing and metabolism based on the database and literature survey. Then, for those genes, we used a population genomics approach based on ten populations over 76,156 human genomes in the gnomAD database. RESULTS: Statistical tests of means and medians of the minor allele frequencies did not show any significant difference among populations. However, we observed substantial differences between two functional groups, glutamate sensing and glutamate metabolism, in populations of Latino/admixed American, Ashkenazi Jewish, and Others. Interestingly, we could find significant differences between the African population and the East Asian population at the single nucleotide polymorphism level of glutamate metabolism genes, but no clear differences were noted in glutamate-sensing genes. These suggest that glutamate-sensing genes are under the functional constraint compared to glutamate metabolism genes. CONCLUSION: Thus, glutamate-sensing genes and metabolism genes have a contrasting mode of the evolution, and glutamate-sensing genes are conservatively evolved, indicating its functional importance.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

Enhancing the sensitivity of bacterial single-cell RNA sequencing using RamDA-seq and Cas9-based rRNA depletion.

Bacterial populations exhibit heterogeneity in gene expression, which facilitates their survival and adaptation to unstable and unpredictable environments through the bet-hedging strategy. However, unraveling the rare subpopulations and heterogeneity in gene expression using population-level gene expression analysis remains a challenging task. Single-cell RNA sequencing (scRNA-seq) has the potential to identify rare subpopulations and capture heterogeneity in bacterial populations, but standard methods for scRNA-seq in bacteria are still under development, mainly due to differences in mRNA abundance and structure between eukaryotic and prokaryotic organisms. In this study, we present a hybrid approach that combines random displacement amplification sequencing (RamDA-seq) with Cas9-based rRNA depletion for scRNA-seq in bacteria. This approach allows cDNA amplification and subsequent sequencing library preparation from low-abundance bacterial RNAs. We evaluated its sequenced read proportion, gene detection sensitivity, and gene expression patterns from the dilution series of total RNA or the sorted single Escherichia coli cells. Our results demonstrated the detection of more than 1000 genes, about 24% of the genes in the E. coli genome, from single cells with less sequencing effort compared to conventional methods. We observed gene expression clusters between different cellular proliferation states or heat shock treatment. The approach demonstrated high detection sensitivity in gene expression analysis compared to current bacterial scRNA-seq methods and proved to be an invaluable tool for understanding the ecology of bacterial populations and capturing the heterogeneity of bacterial gene expression.

Alzheimer's disease heterogeneity explained by polygenic risk scores derived from brain transcriptomic profiles.

INTRODUCTION: Alzheimer's disease (AD) is heterogeneous, both clinically and neuropathologically. We investigated whether polygenic risk scores (PRSs) integrated with transcriptome profiles from AD brains can explain AD clinical heterogeneity. METHODS: We conducted co-expression network analysis and identified gene sets (modules) that were preserved in three AD transcriptome datasets and associated with AD-related neuropathological traits including neuritic plaques (NPs) and neurofibrillary tangles (NFTs). We computed the module-based PRSs (mbPRSs) for each module and tested associations with mbPRSs for cognitive test scores, cognitively defined AD subgroups, and brain imaging data. RESULTS: Of the modules significantly associated with NPs and/or NFTs, the mbPRSs from two modules (M6 and M9) showed distinct associations with language and visuospatial functioning, respectively. They matched clinical subtypes and brain atrophy at specific regions. DISCUSSION: Our findings demonstrate that polygenic profiling based on co-expressed gene sets can explain heterogeneity in AD patients, enabling genetically informed patient stratification and precision medicine in AD. HIGHLIGHTS: Co-expression gene-network analysis in Alzheimer's disease (AD) brains identified gene sets (modules) associated with AD heterogeneity. AD-associated modules were selected when genes in each module were enriched for neuritic plaques and neurofibrillary tangles. Polygenic risk scores from two selected modules were linked to the matching cognitively defined AD subgroups (language and visuospatial subgroups). Polygenic risk scores from the two modules were associated with cognitive performance in language and visuospatial domains and the associations were confirmed in regional-specific brain atrophy data.

Target enrichment of uncultured human oral bacteria with phage-derived molecules found by single-cell genomics.

Advances in culture-independent microbial analysis, such as metagenomics and single-cell genomics, have significantly increased our understanding of microbial lineages. While these methods have uncovered a large number of novel microbial taxa, many remain uncultured, and their function and mode of existence in the environment are still unknown. This study aims to explore the use of bacteriophage-derived molecules as probes for detecting and isolating uncultured bacteria. Here, we proposed multiplex single-cell sequencing to obtain massive uncultured oral bacterial genomes and searched prophage sequences from over 450 obtained human oral bacterial single-amplified genomes (SAGs). The focus was on the cell wall binding domain (CBD) in phage endolysin, and fluorescent protein-fused CBDs were generated based on several CBD gene sequences predicted from Streptococcus SAGs. The ability of the Streptococcus prophage-derived CBDs to detect and enrich specific Streptococcus species from human saliva while maintaining cell viability was confirmed by magnetic separation and flow cytometry. The approach to phage-derived molecule generation based on uncultured bacterial SAG is expected to improve the process of designing molecules that selectively capture or detect specific bacteria, notably from uncultured gram-positive bacteria, and will have applications in isolation and in situ detection of beneficial or pathogenic bacteria.

Single-Cell Level Raman Molecular Profiling Reveals the Classification of Growth Phases of Chaetoceros tenuissimus.

Harmful algal blooms (HABs) are a natural phenomenon caused by outbreaks of algae, resulting in serious problems for aquatic ecosystems and the coastal environment. Chaetoceros tenuissimus (C. tenuissimus) is one of the diatoms responsible for HABs. The growth curve of C. tenuissimus can be observed from beginning to end of HABs: therefore, detailed analysis is necessary to characterize each growth phase of C. tenuissimus. It is important to examine the phenotype of each diatom cell individually, as they display heterogeneity even in the same growth phase. Raman spectroscopy is a label-free technique to elucidate biomolecular profiles and spatial information at the cellular level. Multivariate data analysis (MVA) is an efficient method for the analysis of complicated Raman spectra, to identify molecular features. Here, we utilized Raman microspectroscopy to identify the molecular information of each diatom cell, at the single-cell level. The MVA, together with a support vector machine, which is a machine learning technique, allowed the classification of proliferating and nonproliferating cells. The classification includes polyunsaturated fatty acids such as linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. This study indicated that Raman spectroscopy is an appropriate technique to examine C. tenuissimus at the single-cell level, providing relevant data to assess the correlation between the molecular details obtained from the Raman analysis, at each growth phase.

Combined actions of bacteriophage-encoded genes in Wolbachia-induced male lethality.

Some Wolbachia endosymbionts induce male killing, whereby male offspring of infected females are killed during development; however, the origin and diversity of the underlying mechanisms remain unclear. In this study, we identified a 76 kbp prophage region specific to male-killing Wolbachia hosted by the moth Homona magnanima. The prophage encoded a homolog of the male-killing gene oscar in Ostrinia moths and the wmk gene that induces various toxicities in Drosophila melanogaster. Upon overexpressing these genes in D. melanogaster, wmk-1 and wmk-3 killed all males and most females, whereas Hm-oscar, wmk-2, and wmk-4 had no impact on insect survival. Strikingly, co-expression of tandemly arrayed wmk-3 and wmk-4 killed 90% of males and restored 70% of females, suggesting their conjugated functions for male-specific lethality. While the male-killing gene in the native host remains unknown, our findings highlight the role of bacteriophages in male-killing evolution and differences in male-killing mechanisms among insects.

White spot syndrome virus (WSSV) modulates lipid metabolism in white shrimp.

In addition to the Warburg effect, which increases the availability of energy and biosynthetic building blocks in WSSV-infected shrimp, WSSV also induces both lipolysis at the viral genome replication stage (12 hpi) to provide material and energy for the virus replication, and lipogenesis at the viral late stage (24 hpi) to complete virus morphogenesis by supplying particular species of long-chain fatty acids (LCFAs). Here, we further show that WSSV causes a reduction in lipid droplets (LDs) in hemocytes at the viral genome replication stage, and an increase in LDs in the nuclei of WSSV-infected hemocytes at the viral late stage. In the hepatopancreas, lipolysis is triggered by WSSV infection, and this leads to fatty acids being released into the hemolymph. ß-oxidation inhibition experiment reveals that the fatty acids generated by WSSV-induced lipolysis can be diverted into ß-oxidation for energy production. At the viral late stage, WSSV infection leads to lipogenesis in both the stomach and hepatopancreas, suggesting that fatty acids are in high demand at this stage for virion morphogenesis. Our results demonstrate that WSSV modulates lipid metabolism specifically at different stages to facilitate its replication.

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Linking antigen specific T-cell dynamics in a microfluidic chip to single cell transcription patterns.

A new non-invasive screening profile has been realized that can aid in determining T-cell activation state at single-cell level. Production of activated T-cells with good specificity and stable proliferation is greatly beneficial for advancing adoptive immunotherapy as innate immunological cells are not effective in recognizing and eliminating cancer as expected. The screening method is realized by relating intracellular Ca2+ intensity and motility of T-cells interacting with APC (Antigen Presenting Cells) in a microfluidic chip. The system is tested using APC pulsed with OVA257-264 peptide and its modified affinities (N4, Q4, T4 and V4), and the T-cells from OT-1 mice. In addition, single cell RNA sequencing reveals the activation states of the cells and the clusters from the derived profiles can be indicative of the T-cell activation state. The presented system here can be versatile for a comprehensive application to proceed with T-cell-based immunotherapy and screen the antigen-specific T-cells with excellent efficiency and high proliferation.

Application of organoid culture from HPV18-positive small cell carcinoma of the uterine cervix for precision medicine.

BACKGROUND: Small cell carcinoma of the uterine cervix (SCCC) is a rare and highly malignant human papillomavirus (HPV)-associated cancer in which human genes related to the integration site can serve as a target for precision medicine. The aim of our study was to establish a workflow for precision medicine of HPV-associated cancer using patient-derived organoid. METHODS: Organoid was established from the biopsy of a patient diagnosed with HPV18-positive SCCC. Therapeutic targets were identified by whole exome sequencing (WES) and RNA-seq analysis. Drug sensitivity testing was performed using organoids and organoid-derived mouse xenograft model. RESULTS: WES revealed that both the original tumor and organoid had 19 somatic variants in common, including the KRAS p.G12D pathogenic variant. Meanwhile, RNA-seq revealed that HPV18 was integrated into chromosome 8 at 8q24.21 with increased expression of the proto-oncogene MYC. Drug sensitivity testing revealed that a KRAS pathway inhibitor exerted strong anti-cancer effects on the SCCC organoid compared to a MYC inhibitor, which were also confirmed in the xenograft model. CONCLUSION: In this study, we confirmed two strategies for identifying therapeutic targets of HPV-derived SCCC, WES for identifying pathogenic variants and RNA sequencing for identifying HPV integration sites. Organoid culture is an effective tool for unveiling the oncogenic process of rare tumors and can be a breakthrough for the development of precision medicine for patients with HPV-positive SCCC.

Cells with stem-like properties are associated with the development of HPV18-positive cervical cancer.

The cellular origins of cervical cancer and the histological differentiation of human papillomavirus (HPV)-infected cells remain unexplained. To gain new insights into the carcinogenesis and histological differentiation of HPV-associated cervical cancer, we focused on cervical cancer with mixed histological types. We conducted genomic and transcriptomic analyses of cervical cancers with mixed histological types. The commonality of the cellular origins of these cancers was inferred using phylogenetic analysis and by assessing the HPV integration sites. Carcinogenesis was estimated by analyzing human gene expression profiles in different histological types. Among 42 cervical cancers with known HPV types, mixed histological types were detected in four cases, and three of them were HPV18-positive. Phylogenetic analysis of these three cases revealed that the different histological types had a common cell of origin. Moreover, the HPV-derived transcriptome and HPV integration sites were common among different histological types, suggesting that HPV integration could occur before differentiation into each histological type. Human gene expression profiles indicated that HPV18-positive cancer retained immunologically cold components with stem cell properties. Mixed cervical cancer has a common cellular origin among different histological types, and progenitor cells with stem-like properties may be associated with the development of HPV18-positive cervical cancer.

Protective Role of Endothelial Fibulin-4 in Valvulo-Arterial Integrity.

Background Homeostasis of the vessel wall is cooperatively maintained by endothelial cells (ECs), smooth muscle cells, and adventitial fibroblasts. The genetic deletion of fibulin-4 (Fbln4) in smooth muscle cells (SMKO) leads to the formation of thoracic aortic aneurysms with the disruption of elastic fibers. Although Fbln4 is expressed in the entire vessel wall, its function in ECs and relevance to the maintenance of valvulo-arterial integrity are not fully understood. Methods and Results Gene silencing of FBLN4 was conducted on human aortic ECs to evaluate morphological changes and gene expression profile. Fbln4 double knockout (DKO) mice in ECs and smooth muscle cells were generated and subjected to histological analysis, echocardiography, Western blotting, RNA sequencing, and immunostaining. An evaluation of the thoracic aortic aneurysm phenotype and screening of altered signaling pathways were performed. Knockdown of FBLN4 in human aortic ECs induced mesenchymal cell-like changes with the upregulation of mesenchymal genes, including TAGLN and MYL9. DKO mice showed the exacerbation of thoracic aortic aneurysms when compared with those of SMKO and upregulated Thbs1, a mechanical stress-responsive molecule, throughout the aorta. DKO mice also showed progressive aortic valve thickening with collagen deposition from postnatal day 14, as well as turbulent flow in the ascending aorta. Furthermore, RNA sequencing and immunostaining of the aortic valve revealed the upregulation of genes involved in endothelial-to-mesenchymal transition, inflammatory response, and tissue fibrosis in DKO valves and the presence of activated valve interstitial cells. Conclusions The current study uncovers the pivotal role of endothelial fibulin-4 in the maintenance of valvulo-arterial integrity, which influences thoracic aortic aneurysm progression.

Mycelial differentiation linked avermectin production in Streptomyces avermitilis studied with Raman imaging.

Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolite production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of secondary metabolites at various stages of morphological differentiation. Under solid culture, avermectin was detected in the mycelia formed at the later stages of morphological differentiation (e.g., spore-bearing mycelium and spiral spore chains), but not in the early-stage substrate mycelium. On the contrary, under liquid culture condition, avermectin was found concentrated in the mycelial pellet formed at the early MII stage of differentiation. Furthermore, the chemical profiles of the mycelia were substantially different depending on the culture condition. Raman spectra corresponding to proteins, lipids, and cytochrome were observed in the mycelia irrespective of the stage of morphological differentiation, however, carotenoid was observed under solid culture condition particularly in spore-bearing mycelium and spiral spore chains. KEY POINTS: • Avermectin production is regulated during mycelial differentiation • Liquid and solid culture conditions affects mycelial differentiation • Raman microspectroscopic analysis reveals localization profiles of avermectin.

Direct intracellular detection of biomolecule specific bound-water with Raman spectroscopy.

Lipids, proteins, and nucleic acids have closely associated water molecules (Bound water), which exhibit considerably different physical properties compared to bulk water. Here we investigate the possibility of resolving Raman spectra of the specific hydration shell of these biomolecules in intracellular regions using Raman imaging. Lipids and proteins + nucleic acids Raman spectral components resolved in the analysis showed associated water spectral features, which are uniquely different from that of bulk water. These spectral profiles agree with water spectral profile observed in the case of corresponding hydrated pure biomolecules. The results show the prospects of Raman imaging in examining intracellular hydration in biomolecules and its functional relation.

Direct imaging of intracellular RNA, DNA, and liquid-liquid phase separated membraneless organelles with Raman microspectroscopy.

Methodologies for direct intracellular imaging of RNA and DNA are necessary for the advancement of bioimaging. Here we show direct label-free imaging of RNA and DNA in single cells by isolating their accurate Raman spectra. Raman images of DNA from interphase cells show intact nucleus, while those from mitotic cells reveal condensed chromosome. The condensed chromosome images are accurate enough to assign the stage of mitotic cell division (e.g., metaphase). Raman spectral features indicate B-DNA double helical conformational form in all the cell lines investigated here. The Raman images of RNAs, on the other hand, reveal liquid-liquid phase separated (LLPS) membraneless organelles in interphase cells, which disappears during mitosis. Further, the Raman spectrum of proteins from the intracellular LLPS organelles indicates slight enrichment of amyloid-like secondary structural features. Vibrational imaging of intracellular DNA and RNA simultaneously would open myriad of opportunities for examining functional biochemical aspects of cells and organelles.