Biology subjects

Greene, E.

Publications and source records attributed to Greene, E..

12 recordsLinked to original sources

bioRxiv2026

Bloom-forming bacteria heavily invest in anti-phage defense

Bacterial blooms are characterized by unusually high cell densities and exceptionally low diversity and can profoundly alter ecosystem function and services. Bacteriophages have long been considered an important cause of mortality in blooms, acting as a mechanism for control. Here, we characterize the viral ecology of a long-lasting estuarine bloom of green sulfur bacteria (Chlorobiota). We combined direct cell and viral counts with metagenomic and metaproteomic data to characterize host and phage activity at different time points. The abundance of virus-like particles (VLPs) decreased at high cell densities, suggesting reduced lytic infection rates. The dominant organism, GSB-TRL01 (genus Prosthecochloris), apparently contained a large conjugative plasmid encoding five different anti-phage defense systems. The organism's genome encoded 13 additional defense systems. Compared to the average of five defense systems per microbial genome, this enrichment suggests robust anti-phage defense capabilities. Proteins from ten different defense systems on GSB-TRL01's genome and four systems from the conjugative plasmid were expressed in the proteome. This suggests that GSB-TRL01 invests heavily in anti-phage defense, leading to reduced lysis at high cell densities and allowing blooms to persist for weeks to months. To determine whether this ability is widespread among bloom forming organisms, we compared genomes of putative bloomers to those of non-blooming organisms. We found that bloomer genomes were significantly enriched with anti-phage defense systems. This challenges traditional paradigms of phage ecology in bloom-forming systems and suggests that microbes adapted to high-density growth may have evolved mechanisms to reduce their susceptibility to phage attack.

microbiology↗

bioRxiv2026

Anoxygenic phototrophic Chlorobi use broad metabolic and resource acquisition strategies to support stable near-clonal blooms

Anoxygenic phototrophic green sulfur bacteria (GSB; Chlorobiia) are important primary producers in anoxic and sulfidic environments, whose significance in aquatic ecosystems will expand with coastal deoxygenation. Here, we employed a uniquely comprehensive analytical approach to investigate the formation, maintenance, and collapse of a GSB bloom. We combined multi-omics -- V4V5 and synthetic long read 16S rRNA amplicon sequencing, metagenomics, and metaproteomics -- with total and GSB-specific cell counts, biogeochemical measurements, and isotopic analysis. The GSB bloom exceeded 109 cells ml-1, among the highest environmental cell densities reported to date, with up to 96 % of the bloom consisting of a single strain-level Prosthecochloris lineage (GSB-TRL01). Extreme sulfide concentrations (> 17 mM) coincided with peaking cell density and a shift in isotopic composition. Bloom persistence is supported by tightly coupled sulfur cycling, high rates of nitrogen fixation, mechanisms to tolerate oxidative stress and maintain redox balance, and nutrient acquisition through outer membrane transport systems. A shift in sulfur oxidizing enzymes towards enzymes with higher sulfide affinity proceeded bloom demise. Prosthecochloris GSB-TRL01 differs from closely related lineages in several outer membrane transport systems, including porins to transport phosphate and tonB-dependent transporters. Collectively, these findings identify physiological and metabolic strategies that may enable near-clonal Prosthecochloris populations to attain extraordinary biomass while coupling the sulfur, carbon, and nitrogen cycles in coastal euxinic environments.

microbiology↗

bioRxiv2026

Satellite Glial Cells Drive Homeostatic Synaptic Structural Plasticity in Sympathetic Neurons

Sympathetic neuronal (SN) activity critically regulates the development and function of peripheral organs and tissues. The demonstration of activity-dependent modulation of SN output suggests that compensatory forms of plasticity could contribute to maintaining the stability of sympathetic circuits. Such plasticity mechanisms could act to restrain SN hyperactivity, a key driver of hypertension in humans and in the spontaneously hypertensive rat (SHR). In this study we examined how long-term changes in activity impact synaptic properties in postnatal sympathetic neuron cultures using chemogenetic and pharmacological manipulations and by examining the effects of enhanced activity of SHR neurons. We showed that bidirectional changes in neuronal activity resulted in homeostatic shifts in synaptic density to counteract long-term activity manipulations. In the absence of sympathetic satellite glial cells (SGCs) there was no synaptic compensation in the cultures. Direct chemogenetic activation of SGCs was sufficient to drive a decrease in synaptic sites and neuronal activity, while glial inhibition blocked activity-dependent synaptic compensation, demonstrating a role for the SGCs in homeostatic regulation of synaptic properties. We found that the SGCs responded to cholinergic signaling by downregulating the expression of the synaptic regulators NGF and TNF, suggesting that reciprocal signaling between SNs and SGCs acts to stabilize sympathetic output during long-term changes in circuit activity. Finally, we showed that these plasticity mechanisms are disrupted in postnatal SHR neurons, with an attenuated neuronal response to glia signaling during synapse formation and activity-dependent plasticity. Taken together, this work describes a new homeostatic activity-dependent plasticity mechanism in the peripheral nervous system.

neuroscience↗

bioRxiv2025

Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity

To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 [A] conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

bioinformatics↗

bioRxiv2025

High-Dimensional Protein Analysis Uncovers Distinct Immunological and Stromal Signatures Between Primary and Metastatic Pancreatic Ductal Adenocarcinoma

Our understanding of the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) primarily stems from murine models or primary patient tumors. While metastatic tumors have generally less immune infiltration compared to primary tumors, the specific cellular features of metastatic PDAC remain understudied. This knowledge gap is impactful as most patients present with metastatic disease and are most often enrolled in clinical trials. We hypothesized PDAC tumors harbor distinct immunologic and stromal features depending on their anatomical site. Using multiplex immunohistochemistry (mIHC), spatial analysis, and single-cell mass cytometry (CyTOF), we uncover dominant immune and stromal cell populations in tumors derived from 27 primary and 26 liver metastases. Metastatic liver tumors from PDAC patients contained fewer T cells and alpha-smooth muscle actin (-SMA+) activated fibroblasts than primary lesions, while CD68+ cells were more abundant. Spatial analyses revealed distinct immune cell communities in primary and metastatic PDAC, whereby CK19+ cells clustered differentially with -SMA+, CD3+, and CD68+ cells, depending on tumor site. When comparing tumor-associated regions, the proportion of peritumoral CK19- cells remained consistent, but their composition varied by disease site. CD8+ T cells were significantly less frequent in metastatic tumors, while both CD4+ and CD8+ T cells present in primary tumors expressed more transcription factors (TFs) associated with suppressive properties, including FoxP3 and ROR{gamma}t. CyTOF revealed that T cells co-expressed multiple inhibitory checkpoint receptors, with LAG-3 and PD-1 predominating. This report reveals that primary and metastatic tumors from PDAC patients harbor vastly distinct immunologic and stromal features at the protein level. Statement of SignificanceProtein level analysis reveals distinct immunological and stromal features between primary and metastatic PDAC tumors, offering a rationale for immunotherapies that target myeloid cells and increase T cell abundance in metastatic disease.

cancer biology↗

bioRxiv2024

Lineage-specific amino acids define functional attributes of the protomer-protomer interfaces for the Rad51 and Dmc1 recombinases

Most eukaryotes possess two Rad51/RecA family DNA recombinases that are thought to have arisen from an ancient gene duplication event: Rad51, which is expressed in both mitosis and meiosis; and Dmc1, which is only expressed in meiosis. To explore the evolutionary relationship between these recombinases, here, we present high-resolution CryoEM structures of S. cerevisiae Rad51 filaments and S. cerevisiae Dmc1 filaments bound to ssDNA, which reveal a pair of stacked interfacial aromatic amino acid residues that are nearly universally conserved in Rad51 but are absent from Dmc1. We use a combination of bioinformatics, genetic analysis of natural sequence variation, and deep mutational analysis to probe the functionally tolerated sequence space for these stacked aromatic residues. Our findings demonstrate that the functional landscape of the interfacial aromatic residues within the Rad51 filament is highly constrained. In contrast, the amino acids at the equivalent positions within the Dmc1 filament exhibit a broad functional landscape. This work helps highlight the distinct evolutionary trajectories of these two eukaryotic recombinases, which may have contributed to their functional and mechanistic divergence. AUTHOR SUMMARYMost eukaryotic organisms have two closely related proteins, Rad51 and Dmc1, that are needed for different aspects of genetic recombination. These proteins may have evolved from a single gene that was duplicated during the early evolution of eukaryotes. Rad51 is active during both normal cell division (mitosis) and sexual reproduction (meiosis), while Dmc1 is only active during meiosis. To better understand how these proteins are related, we studied their three-dimensional structures using high resolution cryogenic electron microscopy. Our findings show that Rad51 has a specific set of conserved amino acids located at the protein interfaces, but this set of amino acids is different in Dmc1. We used a series of genetic approaches to analyze how these amino acids affect the proteins function. Our results show that Rad51 has a strict set of rules governing the identify of these amino acids, whereas Dmc1 does not. This research sheds light on how Rad51 and Dmc1 have evolved differently, leading to distinct functions in genetic recombination.

genetics↗

bioRxiv2024

Protein-based cell population discovery and annotation for CITE-seq data identifies cellular phenotypes associated with critical COVID-19 severity

Technologies such as Cellular Indexing of Transcriptomes and Epitopes sequencing (CITE-seq) and RNA Expression and Protein sequencing (REAP-seq) augment unimodal single-cell RNA sequencing (scRNA-seq) by simultaneously measuring expression of cell-surface proteins using antibody derived oligonucleotide tags (ADT). These protocols have been increasingly used to resolve cellular populations that are difficult to infer from gene expression alone, and to interrogate the relationship between gene and protein expression at a single-cell level. However, the ADT-based protein expression component of these assays remains widely underutilized as a primary tool to discover and annotate cell populations, in contrast to flow cytometry which has used surface protein expression in this fashion for decades. Therefore, we hypothesized that computational tools used for flow cytometry data analysis could be harnessed and scaled to analyze ADT data. Here we apply Ozette Discovery, a recently-developed method for flow cytometry analysis, to re-analyze a large (>400,000 cells) published COVID-19 CITE-seq dataset. Using the protein expression data alone, Ozette Discovery is able to identify granular, robust, and interpretable cellular phenotypes in a high-throughput manner. In particular, we identify a population of CLEC12A+CD11b+CD14- myeloid cells that are specifically expanded in patients with critical COVID-19, and can only be resolved by their protein expression profiles. Using the longitudinal gene expression data from this dataset, we find that early expression of interferon response genes precedes the expansion of this subset, and that early expression of PRF1 and GZMB within specific Ozette Discovery phenotypes provides a RNA biomarker of critical COVID-19. In summary, Ozette Discovery demonstrates that taking a protein-centric approach to cell phenotype annotation in CITE-seq data can achieve the potential that dual RNA/protein assays provide in mixed samples: instantaneous in silico flow sorting, and unbiased RNA-seq profiling. HIGHLIGHTSO_LIOzette Discovery provides an alternative method for data-driven annotation of granular and homogeneous cell phenotypes in CITE-seq data using protein expression data alone. C_LIO_LIOur approach inherently accommodates for batch effects, and our novel background-normalization method improves the signal:noise ratio of these notoriously noisy protein measurements. C_LIO_LIWhile these subpopulations are not derived from RNA profiles, they have distinct and interpretable RNA signatures. C_LIO_LIWe find a population of CLEC12A+CD11b+CD14- myeloid cells associated with critical COVID-19 severity that can only be identified by their protein profiles, and identify early expression of interferon response genes in a CD4 T cell subset as a predictor of CLEC12A+CD11b+CD14- cell expansion. C_LIO_LIPeforming differential expression analysis within our identified phenotypes reveals predictors of COVID-19 severity that are not found with coarser annotations. C_LI

bioinformatics↗

bioRxiv2019

Evaluating spatiotemporal integration of shape cues

Prior work has shown that humans can successfully identify letters that are constructed with a sparse array of dots, wherein the dot pattern reflects the strokes that would normally be used to fashion a given letter. In the present work the dots were briefly displayed, one at a time in sequence, varying the spatial order in which they were shown. A forward sequence was spatially ordered as though one were passing a stroke across the dots to connect them. Experiments compared this baseline condition to the following three conditions: a) the dot sequence was spatially ordered, but in the reverse direction from how letter strokes might normally be written; b) the dots in each stroke of the letter were displayed in a random order; c) the sequence of displayed dots were chosen for display from any location in the letter. Significant differences were found between the baseline condition and all three of the comparison conditions, with letter recognition being far worse for the random conditions than for conditions that provided consistent spatial ordering of dot sequences. These findings show that spatial order is critical for integration of shape cues that have been sequentially displayed.

neuroscience↗

bioRxiv2019

Diffuse Reflectance Spectroscopy reveals heat stress-induced changes in hemoglobin profile in chicken breast

Global rise in incidence of woody breast (WB) syndrome imposes a significant economic burden on the poultry industry. The increase in WB is due to the large increase in the weight of chickens these days within a very short period. An early determination of WB can significantly reduce losses to the poultry industry. Diffuse reflectance spectroscopy provides a noninvasive and rapid method to interrogate tissue function. The sensitivity of DRS to the distinct absorption spectra of oxygenated and deoxygenated hemoglobin allows accurate quantification of average hemoglobin concentration and vascular oxygenation within the sampled tissue. In this study, we used diffuse reflectance spectroscopy to monitor breast hemoglobin concentration (THb) and vascular oxygen saturation (sO2) of 16 chickens that were exposed to heat stress (HS). HS is an important cause of WB myopathy in chickens. Animals were exposed to heat-stress (HS) and optical data were acquired at three time points: at baseline prior to heat stress, 2 days, and 21 days after initiation of HS. Our results show that animals from control and HS groups had a steady decay in optically derived breast hemoglobin concentration consistent with independent i-STAT measurements made on blood sampled from the femoral artery and could provide a noninvasive technology for monitoring tissue function in the poultry industry.

bioengineering↗

bioRxiv2019

A new data-driven cell population discovery and annotation method for single-cell data, FAUST, reveals correlates of clinical response to cancer immunotherapy

High-dimensional single-cell cytometry is routinely used to characterize patient responses to cancer immunotherapy and other treatments. This has produced a wealth of datasets ripe for exploration but whose biological and technical heterogeneity make them difficult to analyze with current tools. We introduce a new interpretable machine learning method for single-cell mass and flow cytometry studies, FAUST, that robustly performs unbiased cell population discovery and annotation. FAUST processes data on a per-sample basis and returns biologically interpretable cell phenotypes that can be compared across studies, making it well-suited for the analysis and integration of complex datasets. We demonstrate how FAUST can be used for candidate biomarker discovery and validation by applying it to a flow cytometry dataset from a Merkel cell carcinoma anti-PD-1 trial and discover new CD4+ and CD8+ effector-memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. We then use FAUST to validate these correlates in an independent CyTOF dataset from a published metastatic melanoma trial. Importantly, existing state-of-the-art computational discovery approaches as well as prior manual analysis did not detect these or any other statistically significant T cell sub-populations associated with anti-PD-1 treatment in either data set. We further validate our methodology by using FAUST to replicate the discovery of a previously reported myeloid correlate in a different published melanoma trial, and validate the correlate by identifying it de novo in two additional independent trials. FAUSTs phenotypic annotations can be used to perform cross-study data integration in the presence of heterogeneous data and diverse immunophenotyping staining panels, enabling hypothesis-driven inference about cell sub-population abundance through a multivariate modeling framework we call Phenotypic and Functional Differential Abundance (PFDA). We demonstrate this approach on data from myeloid and T cell panels across multiple trials. Together, these results establish FAUST as a powerful and versatile new approach for unbiased discovery in single-cell cytometry.

bioinformatics↗

bioRxiv2019

Human condensin I and II drive extensive ATP-dependent compaction of nucleosome-bound DNA

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-binding sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome fibers. These observations shed light on critical processes involved in genome organization in human cells.\n\nOne Sentence SummaryATP-dependent DNA compaction by human condensin complexes.

biochemistry↗

bioRxiv2017

The Condensin Complex Is A Mechanochemical Motor That Translocates Along DNA

One Sentence SummarySingle-molecule imaging reveals that eukaryotic condensin is a highly processive DNA-translocating motor complex.\n\nAbstractCondensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. Here, we use single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of ATP hydrolysis-dependent translocation along double-stranded DNA. Condensins translocation activity is rapid and highly processive, with individual complexes traveling an average distance of [≥]10 kilobases at a velocity of [~]60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its [~]50-nanometer coiled-coil subunits, suggestive of a translocation mechanism that is distinct from any reported DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.

biophysics↗