Biology subjects

Han, X.

Publications and source records attributed to Han, X..

At least 217 records · Page 12Linked to original sources

bioRxiv2022

Single and complex spikes relay distinct frequency-dependent circuit information in the hippocampus

Hippocampal neurons generate either single spikes or stereotyped bursts of spikes known as complex spikes. Although single and complex spikes co-occur in the same neuron, their contribution to information processing remains unclear. We analyzed hippocampal CA1 neurons in awake mice and in behaving rats, combining cellular membrane voltage imaging with optogenetics and extracellular recordings. We found that network-driven subthreshold membrane rhythms in the theta versus gamma frequencies preferably entrained complex versus single spikes in individual neurons. Optogenetic membrane perturbation revealed a causal link between subthreshold theta and gamma power and the initiation of complex versus single spikes. Further, single and complex spikes exhibited different place field properties and frequency-dependent coding during spatial navigation. Thus, individual hippocampal neurons do not integrate theta and gamma rhythms into a combined spike timing code, but instead, transmit frequency-specific information as distinct output modes of single versus complex spikes during spatial cognition.

neuroscience↗

bioRxiv2022

Deposit-feeding worms control subsurface ecosystem functioning in intertidal sediment with strong physical forcing

Intertidal sands are global hotspots of terrestrial and marine carbon cycling with strong hydrodynamic forcing by waves and tides and high macrofaunal activity. Yet, the relative importance of hydrodynamics and macrofauna in controlling these ecosystems remains unclear. Here we compare bacterial, archaeal, and eukaryotic communities in upper intertidal sands dominated by subsurface deposit-feeding worms (Abarenicola pacifica) to adjacent worm-free areas. We show that hydrodynamic forcing controls organismal assemblages in surface sediments, while in deeper layers selective feeding by worms on fine, algae-rich particles strongly decreases the abundance and richness of all three domains. In these deeper layers, bacterial and eukaryotic network connectivity decreases, while percentages of taxa involved in degradation of refractory organic macrostructures, oxidative nitrogen and sulfur cycling, and macrofaunal symbioses, increase. Our findings reveal macrofaunal activity as the key driver of ecosystem functioning and carbon cycling in intertidal sands below the mainly physically controlled surface layer. Significance StatementHydrodynamics and bioturbation are the main forces controlling chemical exchanges between sediment and seawater in coastal environments. However, little is known about the relative impact of both processes on sediment biological communities. We show that intertidal sand ecosystems dominated by lugworms can be divided into vertically distinct hydrodynamically and biologically controlled layers. Hydrodynamic forcing controls biological communities in surface layers by regulating organic carbon and electron acceptor inputs. By contrast, lugworms structure subsurface ecosystems through the selective consumption of fine particles, which diminishes microbial and eukaryotic populations and weakens ecological networks, while promoting the burial of, mostly terrestrial, macrodetritus. Our study demonstrates that globally distributed marine invertebrates control intertidal sand ecosystems below the physically controlled surface layer.

microbiology↗

bioRxiv2022

PPAR-γ activation promotes xenogenic bioroot regeneration by attenuating the xenograft induced-oxidative stress

Objectivexenogenic organ transplantation has been considered the most promising strategy in providing possible subtitutes with physiological function of the failing organs as well as solving the problem of insufficient donor sources. However, the xenograft, suffered from immune rejection and ischemia-reperfusion injury (IRI), causes massive ROS expression and the subsequent cell apoptosis, leading to the xenograft failure. Our previous study found a positive role of PPAR-{gamma} in anti-inflammation through its immunomodulation effects, which inspires us to apply PPAR-{gamma} agonist rosiglitazone (RSG) to address survival issue of xenograft with the potential to eliminate the excessive ROS. MethodsXenogenic bioroot was constructed by wrapping the dental follicle cells (DFC) with porcine extracellular matrix (pECM). The hydrogen peroxide (H2O2)-induced DFC was pretreated with RSG to observe its protection on the damaged biological function. Immunoflourescence staining and transmission electron microscope were used to detect the intracellular ROS level. SD rat orthotopic transplantation model and SOD1 knockout mice subcutaneous transplantation model were applied to explore the regenerative outcome of the xenograft. ResultsRSG pretreatment significantly reduced the adverse effects of H2O2 on DFC with decreased intracellular ROS expression and alleviated mitochondrial damage. In vivo results confirmed RSG administration substantially enhanced the hosts antioxidant capacity with reduced osteoclasts formation and increased periodontal ligament like tissue regeneration efficiency, maximumly maintaining the xenograft function. ConclusionsRSG preconditioning could preserve the biological properties of the transplanted stem cells under OS microenvironment and promote organ regeneration by attenuating the inflammatory reaction and OS injury.

cell biology↗

bioRxiv2022

Deep brain stimulation creates information lesion through membrane depolarization

Deep brain stimulation (DBS) is a promising neuromodulation therapy that alters neural activity via intracranial electrical stimulation. However, the neurophysiological mechanisms of DBS remain largely unknown, because of the difficulty of obtaining cellular resolution recordings without electrical interference. Here, we performed high-speed membrane voltage fluorescence imaging of individual hippocampal CA1 neurons during DBS in awake mice. We discovered that DBS, delivered at either 40Hz or 140Hz, reliably depolarizes somatic membrane potentials. Further, DBS enhanced spike rates and paced membrane voltage and spike timing at the stimulation frequency, though more prominent at 40Hz than 140Hz. To determine how DBS induced membrane voltage change impacts neurons ability to process inputs, we optogenetically evoked membrane depolarization. We found that neurons become unreliable in responding to optogenetic inputs during DBS, particularly during 140Hz DBS. These results demonstrate that DBS produces powerful membrane depolarization that interferes with neurons ability to process inputs, creating information lesion.

neuroscience↗

bioRxiv2022

Paradoxical activation of SREBP1c and de novo lipogenesis by hepatocyte-selective ACLY depletion in obese mice.

Hepatic steatosis associated with high fat diets, obesity and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl-coenzyme A (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high fat feeding is unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, while in contrast ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c (SREBP1c) and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels due to its increased conversion to Malonyl CoA (MalCoA) and palmitate. Together, these data indicate that in HFD fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or MalCoA, but rather by activities of DNL enzymes.

molecular biology↗

bioRxiv2022

Incorporating the image formation process into deep learning improves network performance in deconvolution applications

We present Richardson-Lucy Network (RLN), a fast and lightweight deep learning method for 3D fluorescence microscopy deconvolution. RLN combines the traditional Richardson-Lucy iteration with a fully convolutional network structure, improving network interpretability and robustness. Containing only [~]16 thousand parameters, RLN enables 4- to 50-fold faster processing than purely data-driven networks with many more parameters. By visual and quantitative analysis, we show that RLN provides better deconvolution, better generalizability, and fewer artifacts than other networks, especially along the axial dimension. RLN outperforms Richardson-Lucy deconvolution on volumes contaminated with severe out of focus fluorescence or noise and provides 4- to 6-fold faster reconstructions of large, cleared tissue datasets than classic multi-view pipelines. We demonstrate RLNs performance on cells, tissues, and embryos imaged with widefield-, light sheet-, and structured illumination microscopy.

biophysics↗

bioRxiv2022

The ITPRIPL1- CD3ϵ axis: a novel immune checkpoint controlling T cells activation

The immune system is critical to fighting infections and disease. The molecular recognition of harmful entities takes place when antigen-presenting cells (APC) harboring major histocompatibility complex (MHC) molecules bound to peptides derived from harmful antigens (ligand) dock on specific T cell receptor (TCR)-CD3 complex (receptor) at the surface of CD8+ T cells. The discovery of a general immune checkpoint mechanism to avoid the harmful impact of T cell hyperactivation provoked a paradigm shift. The clinical relevance of this mechanism is highlighted by the fact that PD-1 and PD-L1 inhibitors are very effective at boosting immune reactions. Still, immune evasion frequently happens. The observation that some PD-1/PD-L1 negative tumors have a poor immune response opens the door to identifying a novel immune checkpoint mechanism. Here, we discovered that ITPRIPL1, a gene with unknown function, impairs T cell activation. Surprisingly, we found that CD3{varepsilon} is the direct receptor of ITPRIPL1. This novel immune checkpoint was validated as a drug target using ITPRIPL1 KO mice and monoclonal antibodies. Thus, targeting the ITPRIPL1-CD3e axis, especially in PD-1 - PDL-1 negative patients, is a promising therapeutic strategy to reduce immune evasion.

immunology↗

bioRxiv2022

A cooperative switch within the KaiC hexamer revealed by cryo-EM

The circadian clock of cyanobacteria is based on an approximately 24h rhythm in the phosphorylation level of KaiC, a hexameric ATPase. This oscillation can be reconstituted in vitro by incubating three proteins together with ATP. Like all chemical oscillators, this system must include a nonlinear, or switch-like, feedback loop, whose nature has been unclear. Here, by using single particle cryo-EM at near-atomic resolution we identified two major conformational states of KaiC subunits, denoted as the exposed state and the buried state, which may provide a structural basis of how the KaiC hexamer changes its conformation during the (day-night) phosphorylation-dephosphorylation cycle. We classify the abundance and pattern of exposed and buried states within hexamers for more than 160,000 KaiC particles. The statistics of the spatial arrangement of the two states in hexamers can be quantitatively fit by a simple statistical physics model with an interaction energy between neighboring subunits and a local field that depends on phosphorylation state. Our study shows that phosphorylation shifts the probability of each conformation and reveals that there is substantial cooperativity between neighboring subunits, which can allow a KaiC hexamer to respond in an ultrasensitive, switch-like manner to changes in the phosphorylation level.

biophysics↗

bioRxiv2022

Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution

The large-scale fluorescence microscopy has enabled the observation of dynamic physiological activities at the single cellular level across the mouse cortex, such as distributed neuronal population representations. However, video-rate high-resolution microscopy at sophisticated biological surfaces in nature keeps a challenging task for the tradeoff between the speed, resolution, and field of view. Here we propose Spinning Disk Multifocal Microscopy (SDiM) for arbitrarily shaped surfaces, which enables imaging at centimeter field-of-view, micrometer resolution and up to 30 frames per second across the depth range of 450 {micro}m. We apply this technique in various microscopic systems, including customized macroscopic systems and the Real-time Ultra-large-Scale imaging at High resolution macroscopy (RUSH), in both the reflective mode and the fluorescence mode, and in the study of cortex-wide single-neuron imaging and immune cell tracking. SDiM provides an opportunity for studying the cortex-wide multi-scale cellular interactions in biological tissues.

bioengineering↗

bioRxiv2022

The effects of caloric restriction on adipose tissue and metabolic health are sex- and age-dependent

Caloric restriction (CR) is a nutritional intervention that reduces the risk of age-related diseases in numerous species, including humans. CRs metabolic effects, including decreased fat mass and improved insulin sensitivity, play an important role in its broader health benefits. However, the extent and basis of sex differences in CRs health benefits are unknown. We found that 30% CR in young (3-month-old) male mice decreased fat mass and improved glucose tolerance and insulin sensitivity, whereas these effects were blunted or absent in young female mice. Females resistance to fat and weight loss was associated with decreased lipolysis, lower systemic energy expenditure and fatty acid oxidation, and increased postprandial lipogenesis compared to males. Positron emission tomography-computed tomography (PET/CT) with 18F-fluorodeoxyglucose (18F-FDG) showed that peripheral glucose uptake was comparable between sexes. Instead, the sex differences in glucose homeostasis were associated with altered hepatic ceramide content and substrate metabolism: compared to CR males, CR females had lower TCA cycle activity but higher blood ketone concentrations, a marker of hepatic acetyl-CoA content. This suggests that males use hepatic acetyl-CoA for the TCA cycle whereas in females it accumulates, thereby stimulating gluconeogenesis and limiting hypoglycaemia during CR. In aged mice (18-months old), when females are anoestrus, CR decreased fat mass and improved glucose homeostasis to a similar extent in both sexes. Finally, in a cohort of overweight and obese humans CR-induced fat loss was also sex- and age-dependent: younger females (<45 years) resisted fat loss compared to younger males while in older subjects (>45 years) this sex difference was absent. Collectively, these studies identify age-dependent sex differences in the metabolic effects of CR and highlight adipose tissue, the liver and oestrogen as key determinants of CRs metabolic benefits. These findings have important implications for understanding the interplay between diet and health and for maximising the benefits of CR in humans. HIGHLIGHTSO_LICaloric restriction (CR) decreases fat mass and improves glucose homeostasis in young male mice, but young females resist these effects. C_LIO_LICR females resist lipolysis, decrease energy expenditure and increase postprandial lipogenesis more than CR males, explaining how females resist fat loss. C_LIO_LISex differences in glucose homeostasis are associated with altered hepatic metabolism and gluconeogenesis, without marked differences in peripheral glucose uptake. C_LIO_LICRs effects on fat loss and glucose homeostasis are comparable in aged male and female mice, implicating oestrogen as the driver of the sexually dimorphic effects in young mice. C_LIO_LIIn humans, females resist CR-induced fat loss in an age-dependent manner, further supporting the role of oestrogen in the sexually dimorphic effects of CR. C_LI

physiology↗

bioRxiv2022

Comparative miRNA Transcriptomics of Mouse and Macaque Reveals Cytoskeleton is An Inhibitor for C. neoformans Invasion into Brain

Cryptococcal meningoencephalitis is an emerging infection shifted from primarily ART-naive to being ART-experienced HIV/AIDS patients, COVID-19 patients and also in immune competent individuals, mainly caused by the human opportunistic pathogen Cryptococcus neoformans, yet mechanisms of the brain or CNS dissemination remain to elucidate, which is the deadest process for the disease. Meanwhile, illustrations of clinically relevant responses in cryptococcosis were limited, as the low availabilities of clinical samples. In this study, macaque and mouse infection models were employed and miRNA-mRNA transcriptomes were performed and combined, which revealed cytoskeleton, a major feather in HIV/AIDS patients, was a centric pathway regulated in both two infection models. Notably, assays of clinical immune cells confirmed an enhanced "Trojan Horse" in HIV/AIDS patients, which can be shut down by cytoskeleton inhibitors. Furthermore, we identified a novel enhancer for macrophage "Trojan Horse", myocilin, and an enhanced fungal burden was achieved in brains of MYOC transgenic mice. Taking together, this study reveals fundamental roles of cytoskeleton and MYOC in blocking fungal CNS dissemination, which not only helps to understand the high prevalence of cryptococcal meningitis in HIV/AIDS, but also facilitates the development of novel drugs for therapies of meningoencephalitis caused by C. neoformans and other pathogenic microorganisms.

microbiology↗

bioRxiv2022

Building an atlas of transposable elements reveals the extensive roles of young SINE in gene regulation, genetic diversity, and complex traits in pigs

Transposable elements (TEs) are an extensive source of genetic polymorphisms and play an indispensable role in chromatin architecture, transcriptional regulatory networks, and genomic evolution. The pig is an important source of animal protein and serves as a biomedical model for humans, yet the functional role of TEs in pigs and their contributions to complex traits are largely unknown. Here, we built a comprehensive catalog of TEs (n = 3,087,929) in pigs by a newly developed pipeline. Through integrating multi-omics data from 21 tissues, we found that SINEs with different ages were significantly associated with genomic regions with distinct functions across tissues. The majority of young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, the expression of transcripts that were derived from the remaining active young SINEs exhibited strong tissue specificity through cross-examining 3,570 RNA-seq from 79 tissues and cell types. Furthermore, we detected 211,067 polymorphic SINEs (polySINEs) in 374 individuals genome-wide and found that they clearly recapitulated known patterns of population admixture in pigs. Out of them, 340 population-specific polySINEs were associated with local adaptation. Mapping these polySINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs in functional genomics and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.

genomics↗

bioRxiv2022

Rapid deep widefield neuron finder driven by virtual calcium imaging data

Widefield microscope provides optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, calcium imaging at cellular resolution has been mostly contaminated by tissue scattering and background signals, making neuronal activities extraction challenging and time-consuming. Here we present a deep widefield neuron finder (DeepWonder), which is fueled by simulated calcium recordings but effectively works on experimental data with an order of magnitude faster speed and improved inference accuracy than traditional approaches. The efficient DeepWonder accomplished fifty-fold signal-to-background ratio enhancement in processing terabytes-scale cortex-wide recording, with over 14000 neurons extracted in 17 hours in workstation-grade computing resources compared to nearly week-long processing time with previous methods. DeepWonder circumvented the numerous computational resources and could serve as a guideline to massive data processing in widefield neuronal imaging.

bioengineering↗

bioRxiv2022

The Intrinsically Disordered Region of Coronins Fine-tunes Oligomerization and Actin Polymerization

Coronins are highly conserved actin-binding proteins (ABPs) in the eukaryotic kingdom for polymerizing actin cytoskeleton. The biochemical activity of coronins is primarily mediated by the structural N-terminal {beta}-propeller and the C-terminal helical coiled-coil (CC) domains, but less is known about the function of a middle nonconserved region, the "unique region (UR)". The coronin UR is an intrinsically disordered region (IDR). Herein, we demonstrate that the low complexity of the UR is a conserved signature of the coronin protein family, and the UR/IDR exhibits a striking evolutionary correlated pattern associated with sequence length. By analyzing the role of the IDR in coronins via coarse-grained simulations, we reveal that evolutionary selection of IDR length is coupled with the oligomerization of IDR-containing proteins (IDPs) to provide optimal functional output. By integrating biochemical and cell biology experiments and protein engineering, we found that the IDR regulates Crn1 biochemical activity, both in vivo and in vitro, by fine-tuning CC domain oligomerization and maintaining Crn1 in a tetrameric state. The IDR-guided optimization of Crn1 oligomerization is critical for Arp2/3-mediated actin polymerization.

cell biology↗

bioRxiv2021

Reversable deformation of artificial cell colony for muscle behavior mimicry triggered by actin polymerization

The mimicry of living tissues from artificial cells is beneficial to understanding the interaction mechanism among cells, as well as holding great potentials in the tissue engineering field. Self-powered artificial cells capable of reversible deformation are developed by encapsulating living mitochondria, actin proteins, and methylcellulose. Upon the addition of pyruvate molecules, the mitochondria produce ATP molecules as energy sources to trigger the polymerization of actin. ATP molecules were produced by mitochondria (2.76x1010/ml) with the concentrations of 35.8{+/-}3.2 {micro}M, 158.2{+/-}19.3 {micro}M and 200.7{+/-}20.1 M by adding pyruvate molecules with the concentration of 3 M, 12 M and 21 M, respectively. The reversible deformation of artificial cells is experienced with spindle shape resulting from the polymerization of actins to form filaments adjacent to the lipid bilayer, subsequently back to spherical shape resulting from the depolymerization of actin filaments upon laser irradiations. The linear colonies composed of these artificial cells exhibit collective contraction and relaxation behavior to mimic muscle tissues. At the stage of maximum contraction, the long axis of each GUV is in parallel to each other. All colonies are synchronized in the contraction phase. The deformation of each GUV in the colonies is influenced by its adjacent GUVs. The muscle-like artificial cell colonies paved the path to develop sustainably self-powered artificial tissues in the field of tissue engineering.

bioengineering↗

bioRxiv2021

Spatiotemporal dynamics of molecular expression pattern and intercellular interactions in glial scar responding to spinal cord injury

Adult regeneration in spinal cord is poor in mammalian but remarkable in the neonatal mammals and some vertebrates, including fish and salamanders. Increasing evidences basis of this interspecies and ontogeny highlighted the pivotal roles of neuron extrinsic factors-the glial scar, which exert confusing inhibiting or promoting regeneration function, but the spatiotemporal ordering of cellular and molecular events that drive repair processes in scar formation remains poorly understood. Here, we firstly constructed tissue-wide gene expression measurements of mouse spinal cords over the course of scar formation using the spatial transcriptomics (ST) technology in Spinal cord injury (SCI) repair. We analyzed the transcriptomes of nearly 15449 spots from 32 samples and distinguished normal and damage response regions. Compared to histological changes, spatial mapping of differentiation transitions in spinal cord injury site delineated the possible trajectory between subpopulations of fibroblast, glia and immune cell more comprehensively and defined the extent of scar boundary and core more accurately. Locally, we identified gene expression gradients from leading edge to the core of scar areas that allow for re-understanding of the scar microenvironment and found some regulators in special cell types, such as Thbs1 and Col1a2 in macrophage, CD36 and Postn in fibroblast, Plxnb2 and Nxpe3 in microglia, Clu in astrocyte and CD74 in oligodendrocyte. Last, we profiled the bidirectional ligand-receptor interactions at the neighbor cluster boundary, contributing to maintain scar architecture during gliosis and fibrosis, and found GPR37L1_PSAP and GPR37_PSAP were top 2 enriched gene-pairs between microglia and fibroblast or microglia and astrocyte. Together, the establishment of these profiles firstly uncovered scar spatial heterogeneity and lineage trajectory, provide an unbiased view of scar and served as a valuable resource for CNS injury treatment. HighlightsO_LISpatial illustration of gene expression pattern after T10 right lateral hemisection. C_LIO_LISpatial atlas of scar formation by 21 cell types around damaged area. C_LIO_LIThe origin, trajectory reconstruction and functional diversity of cell types in different stages of scar formation. C_LIO_LINovel insights for glial scar boundary and potential benefits for recovery intervention after SCI. C_LI

neuroscience↗

bioRxiv2021

Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

biochemistry↗

bioRxiv2021

Single-cell Transcriptomic Analysis Reveals the Cellular Heterogeneity of Mesenchymal Stem Cells

Ex vivo-expanded mesenchymal stem cells (MSCs) have been demonstrated to be a heterogeneous mixture of cells exhibiting varying proliferative, multipotential, and immunomodulatory capacities. However, the exact characteristics of MSCs remain largely unknown. By single-cell RNA sequencing of 61,296 MSCs derived from bone marrow and Whartons jelly, we revealed five distinct subpopulations. The developmental trajectory of these five MSC subpopulations were mapped, revealing a differentiation path from stem-like active proliferative cells (APCs) to multipotent progenitor cells, followed by the branching into two paths - adipogenesis or osteochondrogenesis - and subsequent differentiation into unipotent prechondrocytes. The stem-like APCs, expressing the perivascular mesodermal progenitor markers CSPG4/MCAM/NES, uniquely exhibited strong proliferation and stemness signatures. Remarkably, the prechondrocyte subpopulation specifically expressed immunomodulatory genes and was able to suppress activated CD3+ T cell proliferation in vitro, supporting the role of this population in immunoregulation. In summary, our analysis mapped the heterogeneous subpopulations of MSCs and identified two subpopulations with potential functions in self-renewal and immunoregulation. Our findings advance the definition of MSCs by identifying the specific functions of its heterogeneous cellular composition, allowing for more specific and effective MSC application through the purification of its functional subpopulations.

cell biology↗