Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting.

Single-cell multiomics provides comprehensive insights into gene regulatory networks, cellular diversity, and temporal dynamics. Here, we introduce nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), an integrated platform that enables global profiling of the transcriptome and proteome from same single cells via RNA sequencing and mass spectrometry-based proteomics, respectively. Benchmarking of nanoSPLITS demonstrates high measurement precision with deep proteomic and transcriptomic profiling of single-cells.

Proteome-Scale Tissue Mapping Using Mass Spectrometry Based on Label-Free and Multiplexed Workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput.

Proteome-scale tissue mapping using mass spectrometry based on label-free and multiplexed workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput.

Cell-type-specific transcriptomics uncovers spatial regulatory networks in bioenergy sorghum stems.

Bioenergy sorghum is a low-input, drought-resilient, deep-rooting annual crop that has high biomass yield potential enabling the sustainable production of biofuels, biopower, and bioproducts. Bioenergy sorghum's 4-5 m stems account for ~80% of the harvested biomass. Stems accumulate high levels of sucrose that could be used to synthesize bioethanol and useful biopolymers if information about cell-type gene expression and regulation in stems was available to enable engineering.

Oligosaccharide production and signaling correlate with delayed flowering in an Arabidopsis genotype grown and selected in high [CO2].

Since industrialization began, atmospheric CO2 ([CO2]) has increased from 270 to 415 ppm and is projected to reach 800-1000 ppm this century. Some Arabidopsis thaliana (Arabidopsis) genotypes delayed flowering in elevated [CO2] relative to current [CO2], while others showed no change or accelerations. To predict genotype-specific flowering behaviors, we must understand the mechanisms driving flowering response to rising [CO2].

Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.

Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection.

A crucial role for dynamic expression of components encoding the negative arm of the circadian clock.

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex.

A crucial role for dynamic expression of components encoding the negative arm of the circadian clock.

In the circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component ( ). FRQ interacts with FRH (FRQ-interacting helicase) and CK-1 forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as , that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex.

Synthetic Soil Aggregates: Bioprinted Habitats for High-Throughput Microbial Metaphenomics.

The dynamics of microbial processes are difficult to study in natural soil, owing to the small spatial scales on which microorganisms operate and to the opacity and chemical complexity of the soil habitat. To circumvent these challenges, we have created a 3D-bioprinted habitat that mimics aspects of natural soil aggregates while providing a chemically defined and translucent alternative culturing method for soil microorganisms. Our Synthetic Soil Aggregates (SSAs) retain the porosity, permeability, and patchy resource distribution of natural soil aggregates-parameters that are expected to influence emergent microbial community interactions.

Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus .

Brown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction.

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip.

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow.

Re-routing of Sugar Catabolism Provides a Better Insight Into Fungal Flexibility in Using Plant Biomass-Derived Monomers as Substrates.

The filamentous ascomycete has received increasing interest as a cell factory, being able to efficiently degrade plant cell wall polysaccharides as well as having an extensive metabolism to convert the released monosaccharides into value added compounds. The pentoses D-xylose and L-arabinose are the most abundant monosaccharides in plant biomass after the hexose D-glucose, being major constituents of xylan, pectin and xyloglucan. In this study, the influence of selected pentose catabolic pathway (PCP) deletion strains on growth on plant biomass and re-routing of sugar catabolism was addressed to gain a better understanding of the flexibility of this fungus in using plant biomass-derived monomers.

Intracellular pathways for lignin catabolism in white-rot fungi.

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy.

Counting mRNA Copies in Intact Bacterial Cells by Fluctuation Localization Imaging-Based Fluorescence In Situ Hybridization (fliFISH).

A method for measuring mRNA copies in intact bacterial cells by fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) is presented. Unlike conventional single-molecule FISH, where the presence of a transcript is determined by fluorescence intensity, fliFISH relies on On-Off duty cycles of photo-switching dyes to set a predetermined threshold for distinguishing true signals from background noise. The method provides a quantitative approach for detecting and counting true mRNA copies and rejecting false signals with high accuracy.

Metabolic Interactions between and Pseudomonas fluorescens under Controlled Iron-Limited Conditions.

Iron (Fe) availability has well-known effects on plant and microbial metabolism, but its effects on interspecies interactions are poorly understood. The purpose of this study was to investigate metabolite exchange between the grass strain Bd21 and the soil bacterium SBW25::gfp/lux (SBW25) during Fe limitation under axenic conditions. We compared the transcriptional profiles and root exudate metabolites of plants grown semihydroponically with and without SBW25 inoculation and Fe amendment.

Colonies of the fungus Aspergillus niger are highly differentiated to adapt to local carbon source variation.

Saprobic fungi, such as Aspergillus niger, grow as colonies consisting of a network of branching and fusing hyphae that are often considered to be relatively uniform entities in which nutrients can freely move through the hyphae. In nature, different parts of a colony are often exposed to different nutrients. We have investigated, using a multi-omics approach, adaptation of A.

Rosette core fungal resistance in Arabidopsis thaliana.

Unlike rosette leaves, the mature Arabidopsis rosette core can display full resistance to Botrytis cinerea revealing the importance for spatial and developmental aspects of plant fungal resistance. Arabidopsis thaliana is a model host to investigate plant defense against fungi. However, many of the reports investigating Arabidopsis fungal defense against the necrotrophic fungus, Botrytis cinerea, utilize rosette leaves as host tissue.

Wetland Sediments Host Diverse Microbial Taxa Capable of Cycling Alcohols.

Alcohols are commonly derived from the degradation of organic matter and yet are rarely measured in environmental samples. Wetlands in the Prairie Pothole Region (PPR) support extremely high methane emissions and the highest sulfate reduction rates reported to date, likely contributing to a significant proportion of organic matter mineralization in this system. While ethanol and isopropanol concentrations up to 4 to 5 mM in PPR wetland pore fluids have been implicated in sustaining these high rates of microbial activity, the mechanisms that support alcohol cycling in this ecosystem are poorly understood.

Reference genes for accurate normalization of gene expression in wood-decomposing fungi.

Wood-decomposing fungi efficiently decompose plant lignocellulose, and there is increasing interest in characterizing and perhaps harnessing the fungal gene regulation strategies that enable wood decomposition. Proper interpretation of these fungal mechanisms relies on accurate quantification of gene expression, demanding reliable internal control genes (ICGs) as references. Commonly used ICGs such as actin, however, fluctuate among wood-decomposing fungi under defined conditions.

Multiple mechanisms drive phage infection efficiency in nearly identical hosts.

Phage-host interactions are critical to ecology, evolution, and biotechnology. Central to those is infection efficiency, which remains poorly understood, particularly in nature. Here we apply genome-wide transcriptomics and proteomics to investigate infection efficiency in nature's own experiment: two nearly identical (genetically and physiologically) Bacteroidetes bacterial strains (host18 and host38) that are genetically intractable, but environmentally important, where phage infection efficiency varies.

Phenotypic responses to interspecies competition and commensalism in a naturally-derived microbial co-culture.

The fundamental question of whether different microbial species will co-exist or compete in a given environment depends on context, composition and environmental constraints. Model microbial systems can yield some general principles related to this question. In this study we employed a naturally occurring co-culture composed of heterotrophic bacteria, Halomonas sp.

Multi-time series RNA-seq analysis of Enterobacter lignolyticus SCF1 during growth in lignin-amended medium.

The production of lignocellulosic-derived biofuels is a highly promising source of alternative energy, but it has been constrained by the lack of a microbial platform capable to efficiently degrade this recalcitrant material and cope with by-products that can be toxic to cells. Species that naturally grow in environments where carbon is mainly available as lignin are promising for finding new ways of removing the lignin that protects cellulose for improved conversion of lignin to fuel precursors. Enterobacter lignolyticus SCF1 is a facultative anaerobic Gammaproteobacteria isolated from tropical rain forest soil collected in El Yunque forest, Puerto Rico under anoxic growth conditions with lignin as sole carbon source.