ABSTRACT
Quantification of measurement uncertainty is crucial for robust scientific inference, yet accurate estimates of this uncertainty remain elusive for ecological measures of diversity. Here, we address this longstanding challenge by deriving a closed-form unbiased estimator for the sampling variance of Simpson's diversity index. In numerical tests the estimator consistently outperforms existing approaches, particularly for applications in which species richness exceeds sample size. We apply the estimator to quantify biodiversity loss in marine ecosystems and to demonstrate ligand-dependent contributions of T-cell-receptor chains to specificity, illustrating its versatility across fields. The novel estimator provides researchers with a reliable method for comparing diversity between samples, essential for quantifying biodiversity trends and making informed conservation decisions.
ABSTRACT
The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.
Subject(s)
COVID-19 , Nasopharynx , SARS-CoV-2 , Single-Cell Analysis , T-Lymphocytes , Humans , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Nasopharynx/virology , Nasopharynx/immunology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Interferons/immunology , Interferons/metabolism , Male , Female , Macrophages/immunology , Macrophages/virology , Virus Replication , Epithelial Cells/virology , Epithelial Cells/immunology , Time Factors , AdultABSTRACT
Endosomes are central protein-sorting stations at the crossroads of numerous membrane trafficking pathways in all eukaryotes. They have a key role in protein homeostasis and cellular signalling and are involved in the pathogenesis of numerous diseases. Endosome-associated protein assemblies or coats collect transmembrane cargo proteins and concentrate them into retrieval domains. These domains can extend into tubular carriers, which then pinch off from the endosomal membrane and deliver the cargoes to appropriate subcellular compartments. Here we discuss novel insights into the structure of a number of tubular membrane coats that mediate the recruitment of cargoes into these carriers, focusing on sorting nexin-based coats such as Retromer, Commander and ESCPE-1. We summarize current and emerging views of how selective tubular endosomal carriers form and detach from endosomes by fission, highlighting structural aspects, conceptual challenges and open questions.
ABSTRACT
Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.
Subject(s)
COVID-19 , SARS-CoV-2 , Single-Cell Analysis , Humans , COVID-19/immunology , COVID-19/pathology , SARS-CoV-2/immunology , Male , Female , Middle Aged , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/genetics , Pulmonary Fibrosis/immunology , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/pathology , CD8-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Lung/immunology , Lung/pathology , Lung/diagnostic imaging , Aged , Adult , Inflammation/immunology , Inflammation/pathology , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/cytology , Memory T Cells/immunology , TranscriptomeABSTRACT
PROPPINs constitute a conserved protein family with multiple members being expressed in many eukaryotes. PROPPINs have mainly been investigated for their role in autophagy, where they co-operate with several core factors for autophagosome formation. Recently, novel functions of these proteins on endo-lysosomal compartments have emerged. PROPPINs support the division of these organelles and the formation of tubulo-vesicular cargo carriers that mediate protein exit from them, such as those generated by the Retromer coat. In both cases, PROPPINs provide membrane fission activity. Integrating information from yeast and human cells this review summarizes the most important molecular features that allow these proteins to facilitate membrane fission and thus provide a critical element to endo-lysosomal protein traffic.
Subject(s)
Autophagy , Endosomes , Lysosomes , Humans , Lysosomes/metabolism , Autophagy/physiology , Endosomes/metabolism , Animals , Protein Transport , Intracellular Membranes/metabolismABSTRACT
Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.
Subject(s)
Cell Differentiation , Neurogenesis , Neurons , RNA Isoforms , Humans , Neurogenesis/genetics , Cell Differentiation/genetics , RNA Isoforms/genetics , RNA Isoforms/metabolism , Neurons/metabolism , Neurons/cytology , Alternative Splicing , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Protein Isoforms/metabolism , Protein Isoforms/genetics , Exons/geneticsABSTRACT
BACKGROUND: Corticosteroids are used for induction of remission in patients with moderately to severely active ulcerative colitis. However, up to one-third of patients fail to this therapy. We investigated if fecal microbial composition or its metabolic capacity are associated with response to systemic corticosteroids. METHODS: In this prospective, multicenter study, patients with active ulcerative colitis (Lichtiger score ≥4) receiving systemic corticosteroids were eligible. Data were assessed and fecal samples collected before and after 4 weeks of treatment. Patients were divided into responders (decrease of Lichtiger Score ≥50%) and nonresponders. The fecal microbiome was assessed by the 16S rRNA gene marker and analyzed with QIIME 2. Microbial metabolic pathways were predicted using parsimonious flux balance analysis. RESULTS: Among 93 included patients, 69 (74%) patients responded to corticosteroids after 4 weeks. At baseline, responders could not be distinguished from nonresponders by microbial diversity and composition, except for a subgroup of biologic-naïve patients. Within 4 weeks of treatment, responders experienced changes in beta diversity with enrichment of ascribed beneficial taxa, including Blautia, Anaerostipes, and Bifidobacterium, as well as an increase in predicted butyrate synthesis. Nonresponders had only minor longitudinal taxonomic changes with a significant increase of Streptococcus salivarius and a microbial composition shifting away from responders. CONCLUSION: Baseline microbial diversity and composition seem to be of limited use to predict response to systemic corticosteroids in active ulcerative colitis. Response is longitudinally associated with restoration of microbial composition and its metabolic capacity.
Subject(s)
Colitis, Ulcerative , Humans , Colitis, Ulcerative/therapy , RNA, Ribosomal, 16S/genetics , Prospective Studies , Feces/microbiology , Adrenal Cortex Hormones/therapeutic use , Treatment OutcomeABSTRACT
Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat. When reconstituted with lipid membranes, using subtomogram averaging we determined tilted Atg18 dimer structures bridging two juxtaposed lipid membranes spaced apart by 80 Å. Moreover, lipid reconstitution experiments further delineate the contributions of Atg18's FRRG motif and the amphipathic helical extension in membrane interaction. The observed structural plasticity of Atg18's oligomeric organization and membrane binding properties provide a molecular framework for the positioning of downstream components of the autophagy machinery.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae Proteins/metabolism , Membrane Proteins/metabolism , Membranes/metabolism , Autophagy-Related Proteins/metabolism , Autophagy , LipidsABSTRACT
Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.
Subject(s)
Diphosphates , Saccharomyces cerevisiae , Animals , Cyclin-Dependent Kinases , Mammals , Phosphates , Saccharomyces cerevisiae/geneticsABSTRACT
Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.
Subject(s)
Nuclear Proteins , Transcription Factors , Humans , Nuclear Proteins/genetics , Transcription Factors/genetics , Regulatory Sequences, Nucleic Acid , RNA Polymerase II/genetics , Transcription, Genetic , Cell Cycle Proteins/geneticsABSTRACT
Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.
Subject(s)
Phosphates , Proteomics , Phosphates/chemistry , Polyphosphates/chemistry , ChemistryABSTRACT
BCRs (Abs) and TCRs (or adaptive immune receptors [AIRs]) are the means by which the adaptive immune system recognizes foreign and self-antigens, playing an integral part in host defense, as well as the emergence of autoimmunity. Importantly, the interaction between AIRs and their cognate Ags defies a simple key-in-lock paradigm and is instead a complex many-to-many mapping between an individual's massively diverse AIR repertoire, and a similarly diverse antigenic space. Understanding how adaptive immunity balances specificity with epitopic coverage is a key challenge for the field, and terms such as broad specificity, cross-reactivity, and polyreactivity remain ill-defined and are used inconsistently. In this Immunology Notes and Resources article, a group of experimental, structural, and computational immunologists define commonly used terms associated with AIR binding, describe methodologies to study these binding modes, as well as highlight the implications of these different binding modes for therapeutic design.
Subject(s)
Antigens , Receptors, Antigen, T-Cell , Receptors, Antigen, B-Cell , Immune System/metabolism , AutoimmunityABSTRACT
BACKGROUND: Among patients with ulcerative colitis, 30-50% receive corticosteroids within the first five years after diagnosis. We aimed to reconsider their effectiveness in the context of the biologic era. METHODS: In this prospective, multicenter study, patients with active ulcerative colitis (Lichtiger score ≥ 4) were eligible if initiating systemic corticosteroids. The primary endpoint was clinical response (decrease in the Lichtiger score of ≥50%) at week 4. Secondary endpoints included combined response defined as clinical response and any reduction in elevated biomarkers (CRP and/or calprotectin). Steroid dependence was assessed after three months. RESULTS: A total of 103 patients were included. Clinical response was achieved by 73% of patients, and combined response by 68%. A total of 15% of patients were steroid-dependent. Activity of colitis did not influence short-term response to treatment but increased the risk for steroid dependence. Biologic-naïve patients responded better than biologic-experienced patients. Past smoking history (OR 5.38 [1.71, 20.1], p = 0.003), hemoglobin levels (OR 0.76 [0.57, 0.99] for higher levels, p = 0.045), and biologic experience (OR 3.30 [1.08, 10.6], p = 0.036) were independently associated with nonresponse. CONCLUSION: Disease activity was not associated with short-term response to systemic corticosteroids but was associated with steroid dependence in patients with active ulcerative colitis. Exposure to biologics negatively affects response rates.
ABSTRACT
T cell responses precede antibody and may provide early control of infection. We analyzed the clonal basis of this rapid response following SARS-COV-2 infection. We applied T cell receptor (TCR) sequencing to define the trajectories of individual T cell clones immediately. In SARS-COV-2 PCR+ individuals, a wave of TCRs strongly but transiently expand, frequently peaking the same week as the first positive PCR test. These expanding TCR CDR3s were enriched for sequences functionally annotated as SARS-COV-2 specific. Epitopes recognized by the expanding TCRs were highly conserved between SARS-COV-2 strains but not with circulating human coronaviruses. Many expanding CDR3s were present at high frequency in pre-pandemic repertoires. Early response TCRs specific for lymphocytic choriomeningitis virus epitopes were also found at high frequency in the preinfection naive repertoire. High-frequency naive precursors may allow the T cell response to respond rapidly during the crucial early phases of acute viral infection.
ABSTRACT
European farm households will face increasingly challenging conditions in the coming decades due to climate change, as the frequency and severity of extreme weather events rise. This study assesses the complex interrelations between external framework conditions such as climate change or adjustments in the agricultural price and subsidy schemes with farmers' decision-making. As social aspects remain understudied drivers for agricultural decisions, we also consider value-based characteristics of farmers as internal factors relevant for decision-making. We integrate individual learning as response to extreme weather events into an agent-based model that simulates farmers' decision-making. We applied the model to a region in Eastern Austria that already experiences water scarcity and increasing drought risk from climate change and simulated three future scenarios to compare the effects of changes in socio-economic and climatic conditions. In a cross-comparison, we then investigated how farmers can navigate these changes through individual adaptation. The agricultural trajectories project a decline of active farms between -27 and -37% accompanied by a reduction of agricultural area between -20 and -30% until 2053. The results show that regardless of the scenario conditions, adaptation through learning moderates the decline in the number of active farms and farmland compared to scenarios without adaptive learning. However, adaptation increases the workload of farmers. This highlights the need for labor support for farms. Supplementary Information: The online version contains supplementary material available at 10.1007/s13593-023-00890-z.
ABSTRACT
Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis.
Subject(s)
Diphosphates , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Diphosphates/metabolism , Biological Transport , Homeostasis , Inositol Phosphates/metabolismABSTRACT
The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (Pi ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Cryoelectron Microscopy , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vacuoles/metabolism , Polyphosphates/metabolismABSTRACT
In this work the nonlinear behavior of layered SAW resonators is studied with the help of Finite Element (FE) computations. The full calculations depend strongly on the availability of accurate tensor data. While there are accurate material data for linear computations, the complete sets of higher-order material constants, needed for nonlinear simulations, are still not available for relevant materials. To overcome this problem, scaling factors were used for each available nonlinear tensor. The approach here considers piezoelectricity, dielectricity, eletrostriction and elasticity constants up to fourth order. These factors act as a phenomenological estimate for incomplete tensor data. Since no set of fourth order material constants for LiTaO3 is available, an isotropic approximation for the fourth order elastic constants was applied. As a result, it was found that the fourth order elastic tensor is dominated by one fourth order Lamé constant. With the help of the FE model, derived in two different, but equivalent ways, we investigate the nonlinear behavior of a SAW resonator with a layered material stack. The focus was set to third order nonlinearity. Accordingly, the modeling approach is validated using measurements of third order effects in test resonators. In addition, the acoustic field distribution is analyzed.
ABSTRACT
Cells stabilize intracellular inorganic phosphate (Pi) to compromise between large biosynthetic needs and detrimental bioenergetic effects of Pi. Pi homeostasis in eukaryotes uses Syg1/Pho81/Xpr1 (SPX) domains, which are receptors for inositol pyrophosphates. We explored how polymerization and storage of Pi in acidocalcisome-like vacuoles supports Saccharomyces cerevisiae metabolism and how these cells recognize Pi scarcity. Whereas Pi starvation affects numerous metabolic pathways, beginning Pi scarcity affects few metabolites. These include inositol pyrophosphates and ATP, a low-affinity substrate for inositol pyrophosphate-synthesizing kinases. Declining ATP and inositol pyrophosphates may thus be indicators of impending Pi limitation. Actual Pi starvation triggers accumulation of the purine synthesis intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which activates Pi-dependent transcription factors. Cells lacking inorganic polyphosphate show Pi starvation features already under Pi-replete conditions, suggesting that vacuolar polyphosphate supplies Pi for metabolism even when Pi is abundant. However, polyphosphate deficiency also generates unique metabolic changes that are not observed in starving wild-type cells. Polyphosphate in acidocalcisome-like vacuoles may hence be more than a global phosphate reserve and channel Pi to preferred cellular processes. IMPORTANCE Cells must strike a delicate balance between the high demand of inorganic phosphate (Pi) for synthesizing nucleic acids and phospholipids and its detrimental bioenergetic effects by reducing the free energy of nucleotide hydrolysis. The latter may stall metabolism. Therefore, microorganisms manage the import and export of phosphate, its conversion into osmotically inactive inorganic polyphosphates, and their storage in dedicated organelles (acidocalcisomes). Here, we provide novel insights into metabolic changes that yeast cells may use to signal declining phosphate availability in the cytosol and differentiate it from actual phosphate starvation. We also analyze the role of acidocalcisome-like organelles in phosphate homeostasis. This study uncovers an unexpected role of the polyphosphate pool in these organelles under phosphate-rich conditions, indicating that its metabolic roles go beyond that of a phosphate reserve for surviving starvation.