SEARCH · bioRxiv Science

Results for “biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources

bioRxiv2024

CADET: Enhanced transcriptome-wide association analyses in admixed samples using eQTL summary data

A transcriptome-wide association study (TWAS) is a popular statistical method for identifying genes whose genetically-regulated expression (GReX) component is associated with a trait of interest. Most TWAS approaches fundamentally assume that the training dataset (used to fit the gene expression prediction model) and testing GWAS dataset are from the same ancestrally homogenous population. If this assumption is violated, studies have shown a marked negative impact on expression prediction accuracy as well as reduced power of the downstream genetrait association test. These issues pose a particular problem for admixed individuals, whose genomes represent a mosaic of multiple continental ancestral segments. To resolve these issues, we present CADET, which enables powerful TWAS of admixed cohorts leveraging the local-ancestry (LA) information of the cohort along with summary-level eQTL data from reference panels of different ancestral groups. CADET combines multiple polygenic risk score models based on the summary-level eQTL reference data to predict LA-aware GReX components in admixed test samples. Using simulated data, we compare the imputation accuracy, power, and type I error rate of our proposed LA-aware approach to LA-unaware methods for performing TWASs. We show that CADET performs optimally in nearly all settings regardless of whether the genetic architecture of gene expression is dependent or independent of ancestry. We further illustrate CADET by performing a TWAS of 29 common blood biochemistry phenotypes within an admixed cohort from the UK Biobank and identify 18 hits unique to our LA-aware strategy with the majority of hits supported by existing GWAS findings.

genetics↗

bioRxiv2024

The nascent RNA labelling compound 5-ethynyl uridine (EU) integrates into DNA in the sea anemone Nematostella vectensis

BackgroundThe detection of de novo synthesized mRNA transcripts is crucial for understanding the regulation of eukaryotic transcription. Using nucleoside or nucleotide analogues to label nascent RNA is potentially jeopardized by the ubiquitous presence of ribonucleotide reductase enzymes (RNRs) that can convert ribonucleotides into 2-deoxyribonucleotides, the building blocks of DNA. Despite this challenge, the uridine analogue 5-ethynyl uridine (EU) has been commercialized and routinely used as specific label for nascent RNAs. Here, we employ confocal imaging, flow cytometry and biochemistry methods to study the specificity of EU to label RNA in six different animal species. ResultsWe demonstrate that EU integrates as expected predominantly into RNA of human embryonic kidney cell line (HEK293), the Drosophila wing disc and the comb jelly Mnemiopsis leidyi. In contrast, we found that EU predominantly labels DNA in the sea anemones Nematostella vectensis and Exaiptasia diaphana, and the polychaete Platynereis dumerilii. In Nematostella, we show that inhibiting RNR by hydroxyurea abolishes cell proliferation and the incorporation of EU into DNA. Alternative compounds for labelling nascent RNA, such as 5-ethynyl cytidine (EC), 5-ethynyl uridine triphosphate (EUTP) or 2-ethynyl adenosine (EA) show similarly low specificity for RNA in Nematostella. ConclusionsOur findings raise concerns about the specificity of ethynylated nucleosides and nucleotides, including EU, to label RNA in some animals. We therefore suggest good practice guidelines for using EU as an RNA labelling tool and discuss pitfalls and indicators that help identifying unintentional DNA labelling.

cell biology↗

bioRxiv2024

Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

AimsPerfluorocarbons (PFCs) are inert, oxygen-rich fluids with applications in neonatal and adult liquid ventilation and potential for innovations related to hypoxic environments, such as under water and for space. Despite promising clinical applications, a laboratory model to comprehensively test PFC implications and predict human airway responses is lacking. We hypothesise that an organotypic airway epithelial cell (AEC) model is needed assess PFC impacts and provide predictive outcomes to inform the in vivo scenario that conventional submerged cultures cannot replicate. This study evaluated PFC exposure in two in vitro systems: a traditional submerged model using 16HBE14o- cells and an air-liquid interface (ALI) model using hSABCi and primary nasal AECs, cultured on transwells to mimic the human airway epithelium. MethodsPFC exposures were conducted at 2, 8, and 24 hours (submerged model) and extended to 72 hours (ALI model). We tracked gross morphological changes via microscopy, quantified apoptosis and autophagy markers through protein biochemistry, and assessed epithelial permeability using trans-epithelial electrical resistance (TEER) and tight junction protein abundance. Statistical analyses included at least three biological replicates per condition. ResultsIn the submerged 16HBE14o- model, PFC exposure led to significant apoptotic changes by 24 hours, with marked autophagic disruption. Nutrient deprivation, confirmed by starvation experiments, was a key driver of cytotoxicity due to media/PFC phase separation. Conversely, hSABCi cells in the ALI model remained viable, with no apoptosis or autophagic disruption over the exposure periods (P > 0.05 vs. control). Similarly, primary nasal AECs showed consistent viability and stability in autophagic and apoptotic markers, indicating a more accurate representation of in vivo conditions. TEER measurements and tight junction protein levels in the ALI model suggested PFC did not compromise epithelial integrity (P > 0.05 vs. control). PBS exposure, included as a liquid control, underscored baseline sensitivity in the primary nAEC model, evidenced by SQSTM1 upregulation and pronounced barrier dysfunction over 24-72 hours (P < 0.05-0.001). These findings underscore the unique biocompatibility of PFC in maintaining cellular integrity, in contrast to the disruptive effects observed with PBS. ConclusionThis is the first study to describe PFC exposure in an organotypic airway model. Results indicate that the ALI model more accurately preserves airway epithelial integrity during PFC exposure than submerged models, which are limited by nutrient depletion effects. The findings support the use of ALI cultures to replicate the human airway architecture for evaluating PFCs biological effects and offer a platform for preclinical applications in respiratory medicine. This organotypic approach may inform future therapeutic and hypoxia-related interventions and contributes significantly to the field by providing a viable model for understanding PFC interactions with airway epithelial cells. We are now trialling PFC emulsions that also contain azithromycin and steroid therapies for extended therapeutic applications. Infographic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/620983v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@a2143aorg.highwire.dtl.DTLVardef@ddf97forg.highwire.dtl.DTLVardef@127045eorg.highwire.dtl.DTLVardef@8fe405_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

CytoSpatio: Learning cell type spatial relationships using multirange, multitype point process models

Recent advances in multiplexed fluorescence imaging have provided new opportunities for deciphering the complex spatial relationships among various cell types across diverse tissues. We introduce CytoSpatio, open-source software that constructs generative, multirange, and multitype point process models that capture interactions among multiple cell types at various distances simultaneously. On analyzing five cell types across five tissues, our software showed consistent spatial relationships within the same tissue type, with certain cell types like proliferating T cells consistently clustering across tissue types. It also revealed that the attraction-repulsion relationships between cell types like B cells and CD4-positive T cells vary with tissue type. CytoSpatio can also generate synthetic tissue structures that preserve the spatial relationships seen in training images, a capability not provided by previous descriptive, motif-based approaches. This potentially allows spatially realistic simulations of how cell relationships affect tissue biochemistry.

bioinformatics↗

bioRxiv2024

A geranylgeranyl reductase homolog required for cholesterol production in Myxococcota

Myxococcota is a phylum of sterol producing bacteria. They exhibit a clade depth for sterol biosynthesis unparalleled in the bacterial domain and produce sterols of a biosynthetic complexity that rivals eukaryotes. Additionally, the sterol biosynthesis pathways found in this phylum have been proposed as a potential source for sterol biosynthesis in the last eukaryotic common ancestor, lending an evolutionary importance to our understanding of this pathway in Myxococcota. However, sterol production has only been characterized in a few species and outstanding questions about the evolutionary history of this pathway remain. Here, we identify two myxobacteria, Minicystis rosea and Sandaracinus amylolyticus, capable of cholesterol biosynthesis. These two myxobacteria possess a cholesterol biosynthesis pathway that differs in both the ordering and enzymes involved in biosynthesis compared to Enhygromyxa salina, a myxobacterium previously demonstrated to produce cholesterol, as well as the canonical pathways found in eukaryotes. We characterize an alternative bacterial reductase responsible for performing C-24 reduction, further delineating bacterial cholesterol production from eukaryotes. Finally, we examine the distribution and phylogenetic relationships of sterol biosynthesis proteins across both cultured and uncultured Myxococcota species, providing evidence for multiple acquisition events and instances of both horizontal and vertical transfer at the family level. Altogether, this work further demonstrates the capacity of myxobacteria to synthesize eukaryotic sterols but with an underlying diversity in the biochemical reactions which govern sterol synthesis, suggesting a complex evolutionary history and refining our understanding of how myxobacterial cholesterol production relates to their eukaryotic counterparts. Significance StatementSterols are essential and ubiquitous lipids in eukaryotes, but their significance in bacteria is less understood. Sterol production in Myxococcota, a phylum of developmentally complex predatory bacteria, has provided insight into novel sterol biochemistry and prompted discussion regarding the evolution of this pathway within both the eukaryotic and bacterial domains. Here, we characterize cholesterol biosynthesis in two myxobacteria, providing evidence for distinct pathway organization and identifying a unique protein responsible for C-24 reduction. We couple these results to phylogenomic analysis of sterol biosynthesis within Myxococcota revealing a complicated evolutionary history marked by vertical and horizontal transfer, suggesting a mosaic acquisition of this pathway in Myxococcota and highlighting the complex role myxobacteria may have had in sterol transfer to eukaryotes.

microbiology↗

bioRxiv2024

A receptor-inactivation model for single-celled habituation in Stentor coeruleus

The single-celled ciliate Stentor coeruleus demonstrates habituation to mechanical stimuli, showing that even single cells can manifest a basic form of learning. Although the ability of Stentor to habituate has been extensively documented, the mechanism of learning is currently not known. Here we take a bottom-up approach and investigate a simple biochemistry-based model based on prior electrophysiological measurements in Stentor along with general properties of receptor molecules. In this model, a mechanoreceptor senses the stimulus and leads to channel opening to change membrane potential, with a sufficient change in polarization triggering an action potential that drives contraction. Receptors that are activated can become internalized, after which they can either be degraded or recycled back to the cell surface. This activity-dependent internalization provides a potential means for the cell to learn. Stochastic simulations of this model confirm that it is capable of showing habituation similar to what is seen in actual Stentor cells, including the lack of dishabituation by strong stimuli and the apparently step-like response of individual cells during habituation. The model also can account for several habituation hallmarks that a previous two-state Markov model could not, namely, the dependence of habituation rate on stimulus magnitude, which had to be added onto the two state model but arises naturally in the receptor inactivation model; the rate of response recovery after cessation of stimulation; the ability of high frequency stimulus sequences to drive faster habituation that results in a lower response probability, and the potentiation of habituation by repeated rounds of training and recovery. The model makes the prediction that application of high force stimuli that do not normally habituate should drive habituation to weaker stimuli due to decrease in the receptor number, which serves as an internal hidden variable. We confirmed this prediction using two new sets of experiments involving alternation of weak and strong stimuli. Furthermore, the model predicts that training with high force stimuli delays response recovery to low force stimuli, which aligns with our new experimental data. The model also predicts subliminal accumulation, wherein continuation of training even after habituation has reached asymptotic levels should lead to delayed response recovery, which was also confirmed by new experiments. The model is unable to account for the phenomenon of rate sensitivity, in which habituation caused by higher frequency stimuli is more easily reversed leading to a frequency dependence of response recovery. Such rate sensitivity has not been reported in Stentor. Here we carried out a new set of experiments which are consistent with the models prediction of the lack of rate sensitivity. This work demonstrates how a simple model can suggest new ways to probe single-cell learning at an experimental level. Finally, we interpret the model in terms of a kernel estimator that the cell may use to guide its decisions about how to response to new stimuli as they arise based on information, or the lack thereof, from past stimuli.

cell biology↗

bioRxiv2024

Surface remodeling and inversion of cell-matrix interactions underlies community recognition and dispersal in Vibrio cholerae biofilms

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. While it is commonly assumed that biofilm-dwelling cells are glued together by the matrix, how the cell-matrix interaction depends on the specific biochemistry of the matrix components and how this interaction varies during biofilm growth remains unclear. Here, we investigated cell-matrix interactions in Vibrio cholerae (Vc), the causative agent of cholera. We combine genetics, microscopy, simulation, and biochemical tools to show that Vc cells are not attractive to Vibrio polysaccharide (VPS), the main matrix component, but they can be bridged with each other and to the VPS network through crosslinking by Bap1. Downregulation of VPS and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results suggest a new conceptual model in understanding the intricate cell-matrix interaction as the major driver for biofilm development, which is potentially generalizable to certain other biofilm-forming species and exopolysaccharides.

biophysics↗

bioRxiv2024

Polysomes and mRNA control the biophysical properties of the eukaryotic cytoplasm

The organization and biophysical properties of the cytoplasm influence all cellular reactions, including molecular interactions and the mobility of biomolecules. It has become clear that the cytoplasm does not behave like a simple fluid but instead is a densely crowded and highly organized environment. Yet, the detailed properties of the cytoplasm, the molecular mechanisms that control them and how they influence the biochemistry of cells remain poorly understood. Here, we investigate the diffusive properties of the cytoplasm in silico and in vivo, employing mRNPs (messenger ribonucleoprotein) and GEM (genetically encoded multimeric) particles as rheological probes in proliferating cells. We demonstrate that cytoplasmic diffusivity increases upon polysome disassembly due to translation inhibition or upon a reduction in mRNA levels. Reducing ribosome concentration by up to 20-25% without a change in polysome levels has no effect in vivo. In addition, we show that upon polysome disassembly, mRNA condensation into P-bodies does not affect cytosolic diffusion in budding yeast. Altogether, our results show that mRNAs and their organization into polysomes control the biophysical properties of the eukaryotic cytoplasm. HighlightsO_LIPolysomes control the biophysical properties of cytoplasm. C_LIO_LImRNP and GEM mobility is enhanced upon translation inhibition that leads to polysome disassembly C_LIO_LIPerturbation of mRNA levels leads to an increase in cytosolic diffusion. C_LI

cell biology↗

bioRxiv2024

Schistosome esophageal gland factor MEG-8.2 drives host cell lysis and interacts with host immune proteins

Schistosomes are blood flukes that ingest large amounts of host blood during their intra-mammalian stage. The ingested blood contains leukocytes that can be harmful, yet the parasites survive inside the host for decades, reflecting superb immune evasion mechanisms that remain poorly understood. Our previous work discovered that FoxA, a forkhead transcription factor, drives the production of the esophageal gland, an anterior digestive organ essential for degrading the ingested leukocytes and for in vivo survival. However, a comprehensive molecular makeup of the esophageal gland remains unclear. Importantly, which of the esophageal gland factors are responsible for degrading the ingested leukocytes, their mechanism of action, and how such a function relates to parasite survival and immune evasion remains unknown. Here, we identify additional esophageal gland genes by taking a comparative transcriptomics approach to identify transcripts altered in foxA knockdown adult schistosomes. A targeted RNAi screen coupled with biochemistry reveals that specific domains of the micro-exon gene MEG-8.2, can drive host cell lysis in a concentration-dependent manner. Using pull-down assays coupled with mass spectrometry, we discover that MEG-8.2 interacts with several host membrane and extracellular proteins that play important roles in activating innate and/or adaptive immunity. Together, our findings suggest a dual role for MEG-8.2 in effectively lysing the ingested cells in the esophageal lumen and interacting with specific host proteins to neutralize or suppress the host immunity. These findings lay an important foundation for exploiting esophageal gland factors to treat schistosomiasis.

microbiology↗

bioRxiv2024

Targeted detection of endogenous LINE-1 proteins and ORF2p interactions

BackgroundBoth the expression and activities of LINE-1 (L1) retrotransposons are known to occur in numerous cell-types and are implicated in pathobiological contexts such as aging-related inflammation, autoimmunity, and in cancers. L1s encode two proteins that are translated from bicistronic transcripts. The translation product of ORF1 (ORF1p) has been robustly detected by immunoassays and shotgun mass spectrometry (MS). Yet, more sensitive detection methods would enhance the use of ORF1p as a clinical biomarker. In contrast, until now, no direct evidence of endogenous L1 ORF2 translation to protein (ORF2p) has been shown. Instead, assays for ORF2p have been limited to ectopic L1 ORF over-expression contexts and to indirect detection of endogenous ORF2p enzymatic activity, such as by the sequencing of de novo genomic insertions. Immunoassays for endogenous ORF2p have been problematic, producing apparent false positives due to cross-reactivities, and shotgun MS has not yielded reliable evidence of ORF2p peptides in biological samples. ResultsHere we present targeted mass spectrometry assays, selected and parallel reaction monitoring (SRM and PRM, respectively) to detect and quantify L1 ORF1p and ORF2p at their endogenous abundances. We were able to quantify ORF1p and ORF2p present in our samples down to a range in the low attomoles. Confident in our ability to affinity enrich ORF2p, we describe an interactome associated with endogenous ORF2-containing macromolecular assemblies. ConclusionThis is the first assay to demonstrate sensitive and robust quantitation of endogenous ORF2p. The ability to assay ORF2p directly and quantitatively will improve our understanding of the developmental and diseased cell states where L1 expression and its activity naturally occur. The ability to simultaneously assay endogenous L1 ORF1p and ORF2p is an important step forward for L1 analytical biochemistry. Endogenous ORF2p interactomes can now be presented with confidence that ORF2p is among the enriched proteins.

molecular biology↗

bioRxiv2024

Single-nuclei sequencing of Moricandia arvensis reveals bundle sheath cell function in the photorespiratory shuttle of C3-C4 intermediate Brassicaceae

Spatially confined gene expression determines cell identity and is fundamental to complex plant traits. In the evolutionary transition from C3 to the more efficient C4 photosynthesis, restricting the glycine decarboxylase reaction to bundle sheath cells initiates a carbon concentrating mechanism via the photorespiratory glycine shuttle. This evolutionary step is generally thought to play an essential role in the progression from ancestral C3 to C4 photosynthesis. Plants operating this shuttle are often referred to as C3-C4 intermediates or C2 species. Within the Brassicaceae family, which includes model plants and crops, such species have evolved independently at least five times. However, research on the biochemistry of C3-C4 intermediates in the Brassicaceae has been limited to a few case studies of differentially localized proteins between mesophyll and bundle sheath cells. Here, we leveraged recent advances in single-cell transcriptome sequencing to better understand how new cellular specialization affects interconnected pathways. We generated a single-nuclei RNA sequencing dataset for Moricandia arvensis, a Brassicaceae with C3-C4 intermediate characteristics, and compared it to a publicly available single-cell transcriptome of leaf tissue of the C3 Arabidopsis thaliana. We independently confirmed the localization of selected photorespiratory proteins by electron microscopy of immunogold-labelled leaf sections. Our analysis revealed the shift in expression of genes directly associated with the photorespiratory glycine decarboxylase reaction, but also of related pathways, such as ammonium assimilation, synthesis of specific amino acids, redox regulation, and transport to the M. arvensis bundle sheath. In contrast, the expression of these genes was not restricted to this cell type in the C3 plant. HighlightSingle-nucleus RNA sequencing of Moricandia arvensis reveals bundle sheath cell-specific expression of photorespiration genes and associated pathways beyond glycine decarboxylase, including ammonium assimilation and redox regulation. This highlights the key role of metabolic compartmentalization in supporting C3-C4 intermediate photosynthesis.

plant biology↗

bioRxiv2024

Leaving Science: Attrition of Biologists in 38 OECD Countries

This study examines biologists leaving science in 38 OECD countries in the past two decades. We use publication metadata from a global bibliometric database (raw Scopus data at the micro-level of individual scientists). In a cohort-based and longitudinal fashion, we follow individual men and women scientists over time, from their first to their last publication (N=86,178). We examine four academic disciplines: AGRI (agricultural and biological sciences), BIO (biochemistry, genetics, and molecular biology), IMMU (immunology and microbiology), and NEURO (neuroscience). We apply survival analysis, conceptualizing scientific life as a sequence of scholarly publishing events. Our Kaplan-Meier survival analysis shows how women disappear from science: in BIO, about 60% are still in science after 5 years, 40% after 10 years, and only 20% by the end of the period examined (i.e., after 19 years). The percentages are substantially higher for men: approximately 70%, 50%, and 30%, respectively. Kaplan-Meier estimations indicate that women in the largest discipline (BIO) are 23.26% more likely to leave science after 10 years and 39.74% more likely to leave science at the end of the study period. Gender difference in attrition, slightly visible after 5 years, increase consistently in later career stages. The probability of surviving for women after 15 years varies considerably, from 47.8% in AGRI to 27.6% in IMMU; for men, the probability is about a fifth higher. Our data show that with the passage of time, women disappear from science in ever-larger proportions compared to men. Gender differences in attrition in the four disciplines have been and continue to be high, but comparing the 2000 and 2010 cohorts, have slightly decreased over time.

scientific communication and education↗

bioRxiv2024

Identification of a Cluster of Benzene Activation Enzymes in a Strictly Anoxic Methanogenic Consortium

The Oil Refinery (OR) consortium is a model methanogenic enrichment culture used to study anaerobic benzene degradation. Over half of the cultures bacterial community consists of two closely related Desulfobacterota strains, designated ORM2a and ORM2b, whose mechanisms of benzene activation are unknown. Two new metagenomes, including a complete circularized metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this cultures proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway, as well as downstream {beta}-oxidation proteins yielding acetate. The most abundant proteins identified mapped to two ORM2a gene clusters of unknown function. Homologous and syntenic gene clusters were identified in genomes of ORM2b and a sulfate-reducing Pelotomaculum that also degrades benzene, as well as in nine contigs assembled from hydrothermal vent metagenomes. Extensive homology and structural predictions suggest that the first cluster - termed the "Magic" gene cluster - encodes for enzymes catalyzing the chemically challenging activation of benzene and subsequent transformation steps yielding benzoyl-CoA. The second ("Nanopod") gene cluster encodes a transmembrane complex that may facilitate benzene transport across the cell membrane. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of strict anaerobes specialized for benzene degradation, which we propose naming "Candidatus Anaerobenzenivorax". IMPORTANCEBenzene is a widespread, persistent and toxic pollutant that can accumulate in anoxic environments such as groundwater and sediments. Despite decades of study, the biochemical mechanisms by which benzene is activated under anaerobic conditions remain unproven. This study provides strong genetic and proteomic evidence for a new class of enzymes that initiate anaerobic benzene activation and proposes a preliminary model for their underlying biochemistry. These findings lay a foundation for future biochemical studies and expand our understanding of how microbes carry out extreme redox chemistry in the absence of oxygen.

microbiology↗

bioRxiv2024

Microbial alginate foraging is conserved in geographically and taxonomically distinct ruminant microbiomes

Seaweed plays a crucial role in carbon cycling and is expected to be a valuable resource for sustainable biomass, with applications in biofuel production, human nutrition, and animal feed. Although seaweed has historically been used as a feed source for livestock grazing near coastlines, the process by which it is digested in the rumen remains unknown. Here, we show how the brown algae Saccharina latissima is catabolized in the rumen ecosystem of two different species using in vivo and in vitro experimental systems. We determined that the ruminal decomposition of alginate, a prominent component of the brown algae cell wall, requires microbial catabolic pathways complete with alginate lyases and transport proteins. Evidence of digestion was obtained through a combination of animal models, bacterial imaging, multilayered meta-omics, and enzyme biochemistry. The evolution of and implications for acquisition of alginate utilization loci within geographically and taxonomically distinct ruminants are considered. Graphical abstractSaccharina latissima is a brown alga commonly found in the North Atlantic, Arctic and Pacific oceans. S. latissima was collected from the west coast and Canada and Norway for microbiome studies. Alginate constitutes a substantial portion of the cell wall of S. latissima (SL), and its digestion requires a specific set of enzymes, alginate lyases. We investigated if and how S. latissima is metabolized in geographically distinct rumen ecosystems through in vivo lamb feeding experiments (2.5 and 5% inclusion, DM basis) and in vitro cattle-based rumen simulation technique, RUSITEC, experiments (up to 50% inclusion). Evidence supporting ruminal degradation of alginate was explored using a combination of multilayered meta-omics, physiology (fluorescently labelled S. latissima hot water extracts (FLA-SLAT)) and biochemical characterization of PL6 alginate lyases. O_FIG O_LINKSMALLFIG WIDTH=161 HEIGHT=200 SRC="FIGDIR/small/628917v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@eebe40org.highwire.dtl.DTLVardef@c6fb8org.highwire.dtl.DTLVardef@7a9b1borg.highwire.dtl.DTLVardef@15d30d7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Cryogenic light microscopy with Angstrom precision deciphers structural conformations of PIEZO1

Despite the impressive progress in molecular biochemistry and biophysics, many questions regarding the conformational states of large (transmembrane) protein complexes persist. In the case of the PIEZO protein, investigations by cryogenic electron microscopy (Cryo-EM) and atomic force microscopy (AFM) have established a symmetric trimer structure with three long-bladed domains in a propeller-like configuration. A transition of PIEZO protein from curved to flat conformation is hypothesized to actuate closed and open channels for the flow of ions. However, conclusive high-resolution data on the molecular organization of PIEZO in its native form are lacking. To address this shortcoming, we exploit single-particle cryogenic light microscopy (spCryo-LM) to decipher the conformational states of the mouse PIEZO1 protein (mPIEZO1) in the cell membrane. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen unroofed cell membranes in and out of a cryostat for super-resolution microscopy at liquid helium temperature. By localizing fluorescent labels placed at the extremities of the three blades with [A]ngstrom precision, we ascertain three configurations of the protein with radii of 6, 12, and 20 nm as projected onto the membrane plane. Our data suggest that in the smallest configuration, the blades form a nano-dome structure that is more strongly curved than previously observed and predicted by AlphaFold-3. In the largest conformation, we believe the structure must fully unbend in an anticlockwise manner to form a flat extended state. We attribute the 12 nm conformation, the most frequently occupied state, to an intermediate state and discuss our results in the context of the findings from other groups. Combination of spCryo-LM and Cryo-EM measurements together with in situ photothermal stimulation promises to provide quantitative insight into the interplay between structure and function of PIEZO and other biomolecular complexes in their native environments.

biophysics↗

bioRxiv2024

Nucleosome Spacing Can Fine-Tune Higher Order Chromatin Assembly

Cellular chromatin displays heterogeneous structure and dynamics, properties that control diverse nuclear processes. Models invoke phase separation of conformational ensembles of chromatin fibers as a mechanism regulating chromatin organization in vivo. Here we combine biochemistry and molecular dynamics simulations to examine, at single base-pair resolution, how nucleosome spacing controls chromatin phase separation. We show that as DNA linkers extend from 25 bp to 30 bp, as examplars of 10N+5 and 10N (integer N) bp lengths, chromatin condensates become less thermodynamically stable and nucleosome mobility increases. Simulations reveal that this is due to trade-offs between inter- and intramolecular nucleosome stacking, favored by rigid 10N+5 and 10N bp linkers, respectively. A remodeler can induce or inhibit phase separation by moving nucleosomes, changing the balance between intra- and intermolecular stacking. The intrinsic phase separation capacity of chromatin enables fine tuning of compaction and dynamics, likely contributing to heterogeneous chromatin organization in vivo.

biophysics↗

bioRxiv2024

Cellular Function of a Biomolecular Condensate Is Determined by Its Ultrastructure

Biomolecular condensates play key roles in the spatiotemporal regulation of cellular processes. Yet, the relationship between atomic features and condensate function remains poorly understood. We studied this relationship using the polar organizing protein Z (PopZ) as a model system, revealing how its material properties and cellular function depend on its ultrastructure. We revealed PopZs hierarchical assembly into a filamentous condensate by integrating cryo-electron tomography, biochemistry, single-molecule techniques, and molecular dynamics simulations. The helical domain drives filamentation and condensation, while the disordered domain inhibits them. Phase-dependent conformational changes prevent interfilament contacts in the dilute phase and expose client binding sites in the dense phase. These findings establish a multiscale framework that links molecular interactions and condensate ultrastructure to macroscopic material properties that drive cellular function.

cell biology↗

bioRxiv2025

Mutant p53R175H-Associated Protection of Ovarian Cancer Precursor Cells from Cisplatin Requires Expression of SLC6A6 Taurine Transporter

Tumor suppressor gene TP53 is ubiquitously mutated in ovarian cancer precursor lesions that undergo pervasive accumulation of DNA damage. Prior literature provides evidence that the expression of mutant p53 protein in epithelial cells is associated with increased survival in response to DNA-damaging treatments. Hence, identification and understanding of the mechanisms that, in response to DNA damage accumulation, support survival of ovarian cancer precursor cells carrying p53 mutations might provide important information about the evolution of the disease. Here we used a combination of OC precursor cell models, biochemistry, microscopy, and flow cytometry to provide evidence that the taurine transporter, the SLC6A6 molecule, contributes to cell protection from DNA-damaging (cisplatin) treatment. We found that expression of mutant p53R175H in OC precursor cells, the fallopian tube non-ciliated epithelial (FNE) cells, induced resistance to the DNA-damaging agent cisplatin. Most importantly, shRNA-mediated targeting of SLC6A6 transcript re-sensitized FNE cells expressing mutant p53R175H to cisplatin treatment. Our studies are consistent with the model that the loss of SLC6A6 alters mechanisms involved in the regulation of cell survival in response to DNA damage.

cancer biology↗