Biology subjects

Sun, B.

Publications and source records attributed to Sun, B..

At least 127 records · Page 7Linked to original sources

bioRxiv2019

Model-based decoupling of evoked and spontaneous neural activity in calcium imaging data

The pattern of neural activity evoked by a stimulus can be substantially affected by ongoing spontaneous activity. Separating these two types of activity is particularly important for calcium imaging data given the slow temporal dynamics of calcium indicators. Here we present a statistical model that decouples stimulus-driven activity from low dimensional spontaneous activity in this case. The model identifies hidden factors giving rise to spontaneous activity while jointly estimating stimulus tuning properties that account for the confounding effects that these factors introduce. By applying our model to data from zebrafish optic tectum and mouse visual cortex, we obtain quantitative measurements of the extent that neurons in each case are driven by evoked activity, spontaneous activity, and their interaction. This broadly applicable model brings new insight into population-level neural activity in single trials without averaging away potentially important information encoded in spontaneous activity.

neuroscience↗

bioRxiv2019

Co-localization and confinement of ecto-nucleotidases modulate extracellular adenosine nucleotide pools

1Nucleotides comprise small molecules that perform critical signaling and energetic roles in biological systems. Of these, the concentrations of adenosine and its derivatives, including adenosine tri-, di-, and mono-phosphate are dynamically controlled in the extracellular-space by diphosphohydrolases and ecto-nucleotidases that rapidly degrade such nucleotides. In many instances, the close coupling between cells such as those in synaptic junctions yields tiny extracellular nanodomains, within which the charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of sequentially-coupled diphosphohydrolase/nucleotidase-catalyzed reactions. To rank the relative importance of these factors, we utilize reaction-diffusion numerical simulations to systematically probe coupled enzyme activity in narrow junctions. We perform these simulations in nanoscale geometries representative of narrow extracellular compartments, within which we localize sequentially- and spatially-coupled enzymes. These enzymes catalyze the conversion of a representative charged substrate such as adenosine triphosphate (ATP) into substrates with different net charges, such as adenosine monophosphate (AMP) and adenosine (Ado). Our modeling approach considers electrostatic interactions of diffusing, charged substrates with extracellular membranes, and coupled enzymes. With this model, we find that 1) Reaction rates exhibited confinement effects, namely reduced reaction rates relative to bulk, that were most pronounced when the enzyme was close to the pore size and 2) The presence of charge on the pore boundary further tunes reaction rates by controlling the pooling of substrate near the reactive protein akin to ions near trans-membrane proteins. These findings suggest how remarkable reaction efficiencies of coupled enzymatic processes can be supported in charged and spatially-confined volumes of extracellular spaces.

biophysics↗

bioRxiv2019

Molecular basis of calmodulin-dependent calcineurin activation

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation by Ca2+ is in part driven by binding of calmodulin (CaM) to a Calmodulin (CaM)-recognition motif within the phosphatases regulatory domain (RD); how-ever, secondary interactions between CaM and the Calcineurin (CaN) regulatory domain may be necessary to fully activate CaN [1]. Specifically, it has been shown that the CaN regulatory domain folds upon CaM binding and that there is a region C-terminal to the canonical CaM-binding region, the distal helix, that assumes an helix fold and contributes to activation [1]. We hypothesized in Dunlap et al [1] that this putative helical distal helix is capable of binding CaM in a region distinct from the canonical CaM binding region (CaMBR) site, whereby CaN is activated. To test this hypothesis, we utilized molecular simulations including replica-exchange molecular dynamics, protein-protein docking and computational mutagene-sis to model distal helix conformations. From these simulations we have isolated a potential binding site on CaM (site D) that facilitates moderate affinity inter-protein interactions that may attenuate CaN auto-inhibition. Further, molecular simulations of the distal helix A454E mutation demonstrated weakened distal helix/CaM interactions that were previously shown to impair CaN activity. K30E and G40D mutations of CaM at site D presented similar de-creases in binding affinity predicted by simulations. The prediction was correlated with a phosphatase assay in which these two mutants show reduced CaN activity. This study there-fore provides a potential structural basis for the role of secondary CaM/CaN interactions in mediating CaN activation.

molecular biology↗

bioRxiv2019

Intracellular XBP1-IL-24 axis dismantles cytotoxic unfolded protein response in the liver

Endoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an anti-apoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2 (eIF2) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.

cell biology↗

bioRxiv2019

Century long fertilization reduces stochasticity controlling grassland microbial community succession

Determining the drivers underlying ecological succession is a fundamental goal of ecological research and essential for predicting ecosystem functioning in response to human-induced environmental changes. Although various studies have examined the impacts of nitrogen (N) addition on plant and microbial community diversity, structure and activities, it remains unknown how long-term anthropogenic fertilization affects the ecological succession of microbial functional guilds and its underlying community assembly mechanisms. Here, using archived soils, we examined more than a centurys succession in soil microbial functional communities (from 1870 to 2008) from the Park Grass Experiment at Rothamsted Experimental Station, the longest running ecological experiment in the world. Long-term fertilization was found to significantly alter soil functional community structure and led to increasingly convergent succession of soil microbial communities. Meta-analysis indicated that microbial temporal turnover (w) was highly time scale-dependent, and the w value threshold was estimated as 0.0025 with a threshold time point of approximately 160 years. In addition, the importance of stochastic assembly varied greatly in regulating the succession of different microbial guilds. Fertilization had large to medium effects on reducing ecological stochasticity for microbial guilds involved in carbon (C) fixation and degradation, N fixation and mineralization, and denitrification. This century long-term study elucidated the differing influences of assembly mechanisms on soil microbial functional communities involved in C and N cycling, which could not be derived from taxonomic or phylogenetic approaches.

ecology↗

bioRxiv2019

AP-1 Imprints a Reversible Transcriptional Program of Senescent Cells

Senescent cells play important physiological- and pathophysiological roles in tumor suppression, tissue regeneration, and aging. While select genetic and epigenetic elements crucial for senescence induction were identified, the dynamics, underlying epigenetic mechanisms, and regulatory networks defining senescence competence, induction and maintenance remain poorly understood, precluding a deliberate therapeutic manipulation of these dynamic processes. Here, we show, using dynamic analyses of transcriptome and epigenome profiles, that the epigenetic state of enhancers predetermines their sequential activation during senescence. We demonstrate that activator protein 1 (AP-1) imprints the senescence enhancer landscape effectively regulating transcriptional activities pertinent to the timely execution of the senescence program. We define and validate a hierarchical transcription factor (TF) network model and demonstrate its effectiveness for the design of senescence reprogramming experiments. Together, our findings define the dynamic nature and organizational principles of gene-regulatory elements driving the senescence program and reveal promising inroads for therapeutic manipulation of senescent cells.

genomics↗

bioRxiv2019

Functional arrays of human pluripotent stem cell-derived cardiac microtissues

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be amenable to standard multiwell-plate manipulations and be capable of quantifying tissue-level functions such as contractile force. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom printed multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a ramp. The wells are seeded with cell-laden collagen which, in response to the slope of the ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells and we used it to determine an optimal tissue formulation. We validated contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

bioengineering↗

bioRxiv2018

Cation binding to SERCA

Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca2+) binds SERCA with micro-molar affinity, magnesium (Mg2+) and potassium (K+) also compete with calcium (Ca2+) binding. However, the molecular bases for these competing ions influence on SERCA function and the selectivity of the pump for Ca2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg2+, Ca2+and K+-bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MD-resolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca2+and to a lesser extent for Mg2+ and K+, as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg2+ precludes favorable Mg2+ binding relative to Ca2+, while K+ has limited capacity to bind site I, c) Mg2+ most likely acts as inhibitor and K+ as intermediate in SERCAs reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K+, Ca2+ and Mg2+ concentrations.

biochemistry↗

bioRxiv2018

Seedling leaves allocate lower fractions of nitrogen to photosynthetic apparatus in nitrogen fixing trees (Dalbergia odorifera and Erythrophleum fordii) than in non-nitrogen fixing trees (Betula alnoides and Castanopsis hystrix) in subtropical China

Photosynthetic-nitrogen use efficiency (PNUE) is a useful trait to characterize leaf economics, physiology, and strategy. In this study, we investigated the differences in PNUE, leaf nitrogen (N) allocation, and mesophyll conductance (gm) in Dalbergia odorifera and Erythrophleum fordii (N-fixing trees), and Betula alnoides and Castanopsis hystrix (non-N-fixing trees). Seedlings of the four species were cultured in pots and received the same nutrient solution, water volume, and light. LiCor-6400 was used to determine fluorescence yield, photosynthetic response to light, and intercellular CO2 concentration (Ci). N allocation fractions in the photosynthetic apparatus were calculated according to Niinemets and Tenhunen method; gm was calculated according to variable J, EDO, and A-Ci curve fitting methods. PNUE of D. odorifera and E. fordii were significantly lower than those of B. alnoides and C. hystrix because of their allocation of a lower fraction of leaf N to Rubisco (PR) and bioenergetics (PB). Mesophyll conductance had a significant positive correlation with PNUE in D. odorifera, E. fordii, and B. alnoides. The fraction of leaf N to cell wall (PCW) had a significant negative correlation with PR in B. alnoides and C. hystrix. We conclude that B. alnoides and C. hystrix optimized their leaf N allocation toward photosynthesis, with the trade-off being N allocation to the cell wall and Rubisco. Thus, these two species may have a higher competitive ability in natural ecosystems with fertile soil.

ecology↗

bioRxiv2018

Multiplexed Profiling of Single-cell Extracellular Vesicles Secretion

Extracellular vesicles (EVs) are important intercellular mediators regulating health and disease. Conventional EVs surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EVs secretion. Herein, by using spatially patterned antibodies barcode, we realized multiplexed profiling of single-cell EVs secretion from more than 1000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to deep understanding of previously undifferentiated single cell heterogeneity underlying EVs secretion. Notably, we observed the decrement of certain EV phenotypes (e.g. CD63+EVs) were associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EVs secretion and cytokines secretion simultaneously from the same single cells to investigate multidimensional spectrum of intercellular communications, from which we resolved three functional subgroups with distinct secretion profiles by visualized clustering. In particular, we found EVs secretion and cytokines secretion were generally dominated by different cell subgroups. The technology introduced here enables comprehensive evaluation of EVs secretion heterogeneity at single cell level, which may become an indispensable tool to complement current single cell analysis and EV research.\n\nSignificanceExtracellular vesicles (EVs) are cell derived nano-sized particles medicating cell-cell communication and transferring biology information molecules like nucleic acids to regulate human health and disease. Conventional methods for EV surface markers profiling cant tell the differences in the quantity and phenotypes of EVs secretion between cells. To address this need, we developed a platform for profiling an array of surface markers on EVs from large numbers of single cells, enabling more comprehensive monitoring of cellular communications. Single cell EVs secretion assay led to previously unobserved cell heterogeneity underlying EVs secretion, which might open up new avenues for studying cell communication and cell microenvironment in both basic and clinical research.

bioengineering↗

bioRxiv2018

Acetic acid activates distinct taste pathways in Drosophila to elicit opposing, state-dependent feeding responses

Taste circuits are genetically determined to elicit an innate appetitive or aversive response, ensuring that animals consume nutritious foods and avoid the ingestion of toxins. We have examined the response of the fruit fly Drosophila melanogaster to acetic acid, a tastant that can be a metabolic resource but can also be toxic to the fly. Our data reveal that flies accommodate these conflicting attributes of acetic acid by virtue of a hunger-dependent switch in their behavioral response to this stimulus. Fed flies show taste aversion to acetic acid, likely a response to its potential toxicity, whereas starved flies show a robust appetitive response that may reflect their overriding need for calories. These opposing responses are mediated by two different classes of taste neurons. Acetic acid activates both the sugar and bitter pathways, which have opposing effects on feeding behavior. Hunger shifts the response from aversion to attraction by enhancing the appetitive sugar pathway as well as suppressing the aversive bitter pathway. Thus a single tastant can drive opposing behaviors by activating distinct taste pathways modulated by internal state.

neuroscience↗

bioRxiv2018

Transcriptional trajectories of human kidney disease progression

Our molecular understanding of clinical conditions progressing from acute organ injury to irreversible dysfunction is limited. We used renal transplantation as a model to characterize the transcriptional response along the transition from acute kidney injury to allograft fibrosis in humans. The integrated analysis of 163 transcriptomes with machine learning techniques identified shared and divergent transcriptional trajectories determining distinct clinical outcomes in a heterogeneous population. The molecular map of renal responses to injury was validated in a mouse ischemia-reperfusion injury model and highlighted early markers of disease progression. This generally applicable approach opens the way for an unbiased analysis of progressive diseases.

pathology↗

bioRxiv2018

Late B lymphocyte action in dysfunctional tissue repair following kidney injury and transplantation

The mechanisms initiating the late immune response to allografts are poorly understood. Through transcriptome analysis of serial protocol biopsies in kidney transplant recipients, we found a tight correlation between the initial response to kidney injury and a late B lymphocyte signature associated with renal dysfunction and fibrosis, suggesting a link between dysfunctional repair and immunoreactivity. To specifically investigate the immunological consequences of dysfunctional repair, we followed the mouse kidney up to 18 months after ischemia/reperfusion. Even in the absence of foreign antigens we identified a sustained immune response in conjunction with the transition to chronic kidney damage. This tissue-driven immunological process involved both the innate and the adaptive immune system and eventually induced an antigen-driven proliferation, selection and maturation of B lymphocytes into broadly-reacting antibody secreting cells. These findings reveal an unappreciated role of dysfunctional tissue repair on local immunoregulation with a particular relevance for late transplantation immunobiology.

immunology↗

bioRxiv2018

Helicase Promotes Replication Re-initiation from an RNA Transcript

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind the DNA, resulting in a gap in the newly replicated DNA. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances the fork, the DNAP also moves forward processively, via its association with the helicase. The presence of the DNAP, in turn, increases both helicases processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicases novel role in replication re-initiation, independent of replication restart proteins or primase.

biophysics↗

bioRxiv2018

Dual roles of electrostatic-steering and conformational dynamics in the binding of calcineurin’s intrinsically-disordered recognition domain to calmodulin

calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The CaN regulatory domain (RD) is responsible for regulating the enzymes phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorderto-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating CaM binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder cofacilitate or hinder CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.

biophysics↗

bioRxiv2018

Reversion of the photoperiod dependence of flowering in rice with synthetic Hd1-microProteins

Rice (Oryza sativa) is a facultative short-day plant that flowers very late when grown in non-inductive long day conditions. Photoperiod-dependent flowering in rice is regulated by heading date (Hd1) which acts as both an activator and repressor of flowering in a day length-dependent manner. In order to regulate flowering of rice in long days (LD), overexpression of a synthetic Hd1miP, which is capable of interacting with Hd1, was employed. Transgenic Hd1miP rice plants flowered significantly earlier when grown in LD compared to SD, showing that synthetic microProteins can be used to revert short-day plants to long-day plants. Yield analysis revealed that although the OX-Hd1miP grains are comparable to WT in terms of the size of the grains produced, OX-Hd1miP plants like hd1 knockout plants, are compromised in the number of grains produced and the grain maturity rate, suggesting an additional unrecognized role of Hd1 in grain maturity.

plant biology↗

bioRxiv2018

Dissection of the microProtein miP1 floral repressor complex in Arabidopsis

MicroProteins have emerged as potent regulators of transcription factor activity. Here we use a combination of forward genetics and proteomics to dissect the miP1a/b microProtein complex that acts to delay the floral transition in Arabidopsis. The microProteins miP1a and miP1b can bridge an interaction between the flowering promoting factor CONSTANS (CO) and the TOPLESS (TPL) co-repressor protein to represses flowering. We find that the JUMONJI14 (JMJ14) histone demethylase is part of this repressor complex that can initiate chromatin changes in FLOWERING LOCUS T (FT) gene, the direct target of CO. Plants with mutations in JMJ14 exhibit an early flowering phenotype that is largely dependent on the activity of CO, supporting a role for CO in this repressive complex. When mis-expressed at the shoot apex, CO can induce early flowering only in the jmj14 background. Our results indicate that the repressor acts in the shoot apical meristem to keep it in an undifferentiated state until the leaf-derived florigen signal induces the conversion into a floral meristem.

plant biology↗

bioRxiv2017

ProGeM: A framework for the prioritisation of candidate causal genes at molecular quantitative trait loci

Quantitative trait locus (QTL) mapping of molecular phenotypes such as metabolites, lipids, and proteins through genome-wide association studies (GWAS) represents a powerful means of highlighting molecular mechanisms relevant to human diseases. However, a major challenge of this approach is to identify the causal gene(s) at the observed QTLs. Here we present a framework for the \"Prioritisation of candidate causal Genes at Molecular QTLs\" (ProGeM), which incorporates biological domain-specific annotation data alongside genome annotation data from multiple repositories. We assessed the performance of ProGeM using a reference set of 227 previously reported and extensively curated metabolite QTLs. For 98% of these loci, the expert-curated gene was one of the candidate causal genes prioritised by ProGeM. Benchmarking analyses revealed that 69% of the causal candidates were nearest to the sentinel variant at the investigated molecular QTLs, indicating that genomic proximity is the most reliable indicator of \"true positive\" causal genes. In contrast, cis-gene expression QTL data led to three false positive candidate causal gene assignments for every one true positive assignment. We provide evidence that these conclusions also apply to other molecular phenotypes, suggesting that ProGeM is a powerful and versatile tool for annotating molecular QTLs. ProGeM is freely available via GitHub.

bioinformatics↗