Biology subjects

Lee, J.

Publications and source records attributed to Lee, J..

At least 829 records · Page 46Linked to original sources

bioRxiv2020

Memory reconsolidation impairments in sign-tracking to an audiovisual compound stimulus

Studies of memory reconsolidation of pavlovian memories have typically employed unimodal conditioned stimuli, despite the use of multimodal compound stimuli in other settings. Here we studied sign-tracking behaviour to a compound audiovisual stimulus. First, we observed not unexpectedly that sign-tracking was poorer to the audiovisual compound than to unimodal visual stimuli. Then, we showed that, depending on the parameters of compound stimulus re-exposure at memory reactivation, systemic MK-801 treatment either impaired extinction to improve signtracking at test, or disrupted reconsolidation to impair test behaviour. When memory reactivation consisted of re-exposure to only the auditory component of the compound stimulus, we observed sign-tracking impairments following MK-801 treatment, but only under certain test conditions. This was in contrast to the consistent impairment following reactivation with the full audiovisual compound. Moreover, the parameters of auditory stimulus presentation to enable MK-801-induced impairment at test varied depending on whether the stimulus was presented within or outside the training context. These findings suggest that behaviour under the control of appetitive pavlovian compound stimuli can be modulated by targeting both extinction and reconsolidation, and that it is not necessary to re-expose to the full compound stimulus in order to achieve a degree of modulation of behaviour.

neuroscience↗

bioRxiv2020

Synthesis of diagnostic quality cancer pathology images

Deep learning-based computer vision methods have recently made remarkable breakthroughs in the analysis and classification of cancer pathology images. However, there has been relatively little investigation of the utility of deep neural networks to synthesize medical images. In this study, we evaluated the efficacy of generative adversarial networks (GANs) to synthesize high resolution pathology images of ten histological types of cancer, including five cancer types from The Cancer Genome Atlas (TCGA) and the five major histological subtypes of ovarian carcinoma. The quality of these images was assessed using a comprehensive survey of board-certified pathologists (n = 9) and pathology trainees (n = 6). Our results show that the real and synthetic images are classified by histotype with comparable accuracies, and the synthetic images are visually indistinguishable from real images. Furthermore, we trained deep convolutional neural networks (CNNs) to diagnose the different cancer types and determined that the synthetic images perform as well as additional real images when used to supplement a small training set. These findings have important applications in proficiency testing of medical practitioners and quality assurance in clinical laboratories. Furthermore, training of computer-aided diagnostic systems can benefit from synthetic images where labeled datasets are limited (e.g., rare cancers). We have created a publicly available website where clinicians and researchers can attempt questions from the image survey at http://gan.aimlab.ca/.

bioinformatics↗

bioRxiv2020

Inpainting as a Technique for Estimation of Missing Voxels in Chemical Shift Imaging

Issues with model fitting (i.e. suboptimal standard deviation, linewidth/full-width-at-half-maximum, and/or signal-to-noise ratio) in multi-voxel MRI spectroscopy, or chemical shift imaging (CSI), can result in the significant loss of usable voxels. A potential solution to minimize this problem is to estimate the value of unusable voxels by utilizing information from reliable voxels in the same image. We assessed an image restoration method called inpainting as a tool to restore unusable voxels and compared it with traditional interpolation methods (nearest neighbor, trilinear interpolation and tricubic interpolation). We applied these techniques to N-acetylaspartate (NAA) spectroscopy maps from a CSI dataset. Inpainting exhibited superior performance (lower normalized root-mean-square errors, NRMSE) compared to all other methods considered (ps<0.001). Inpainting maintained its superiority whether the previously unusable voxels were randomly distributed or located in regions most commonly affected by voxel loss in real-world data. Clinical RelevanceThe presence of missing voxels can be problematic, particularly when data are analyzed in standard space, given that only voxels that are contributed to by all participants can be interrogated in these analyses. Inpainting is a promising approach for recovering unusable or missing voxels in voxelwise analyses, particularly in imaging modalities characterized by low SNR such as CSI.

bioengineering↗

bioRxiv2020

A Myosin-7B dependent endocytosis pathway mediates cellular entry of α-Synuclein fibrils and polycation-bearing cargos

Cell-to-cell transmission of misfolding-prone -Synuclein (-Syn) has emerged as a key pathological event in Parkinsons disease. This process is initiated when -Syn-bearing fibrils enter cells via clathrin-mediated endocytosis, but the underlying mechanisms are unclear. Using a CRISPR-mediated knockout screen, we identify SLC35B2 and Myosin-7B (MYO7B) as critical endocytosis regulators for -Syn preformed fibrils (PFF). We show that SLC35B2, as a key regulator of heparan sulfate proteoglycan (HSPG) biosynthesis, is essential for recruiting -Syn PFF to the cell surface because this process is mediated by interactions between negatively charged sugar moieties of HSPGs and clustered K-T-K motifs in -Syn PFF. By contrast, MYO7B regulates -Syn PFF entry by maintaining a plasma-membrane-associated actin network that controls membrane dynamics. Without MYO7B or actin filaments, many clathrin-coated pits fail to be severed from the membrane, causing accumulation of large clathrin-containing scars on the cell surface. Intriguingly, the requirement for MYO7B in endocytosis is only restricted to -Syn PFF and other cargos that enter cells via HSPGs. Thus, by identifying new regulatory factors for -Syn PFF endocytosis, our study defines a mechanistically distinct clathrin-mediated endocytosis pathway that requires additional force generated by MYO7B and actin filaments. SignificanceThe spreading of misfolded protein aggregates such as -Synuclein preformed fibrils (-Syn PFF) from cell to cell is a pathologic hallmark associated with the progression of many neurodegenerative diseases, but it is unclear how mammalian cells take up these large protein aggregates to initiate this prion-like protein transmission process. Here we define the mechanism of -Syn PFF endocytosis using a combination of genetic, biochemical, and live-cell imaging techniques. Our study reveals how -Syn PFF binds to the cell surface heparan sulfate proteoglycans using two lysine-bearing motifs and then enters cells following a Myosin-7B- and actin-dependent endocytosis mechanism that is specifically tailored for polycation-bearing cargos.

cell biology↗

bioRxiv2020

Activation of Rab11a and endocytosis by Phosphatidylinositol 4-kinase III beta promotes oncogenic signaling in breast cancer

Endosomes are now recognized as important sites for regulating signal transduction. Here we show that the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIII{beta}) regulates both endocytic kinetics and receptor signaling in breast cancer cells. PI4KIII{beta} generates phosphatidylinositol 4-phosphate from phosphatidylinositol and is highly expressed in a subset of breast cancers. However, the molecular mechanism by which PI4KIII{beta} promotes breast cancer is unclear. We demonstrate that ectopic PI4KIII{beta} expression increases the rates of both endocytic internalization and recycling. PI4KIII{beta} deletion reduces endocytic kinetics accompanied by a concomitant decrease in activity of the Rab11a GTPase, a protein required for endocytic function. Finally, we find that PI4KIII{beta} activates IGF-IR{beta} signaling dependent on endosome function. Regulation of endocytic function by PI4KIII{beta} is independent of its kinase activity but requires interaction with the Rab11a. This suggests that PI4KIII{beta} controls endosomal kinetics and signaling by directly modulating Rab11a function. Our work suggests a novel regulatory role for PI4KIII{beta} in endosome function and plasma membrane receptor signaling.

cancer biology↗

bioRxiv2020

Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

microbiology↗

bioRxiv2020

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

physiology↗

bioRxiv2020

Biomechanical Regulation of Breast Cancer Metastasis and Progression

Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3K{gamma} inhibitor.

cancer biology↗

bioRxiv2020

Coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes is assisted by CLPP2 in Arabidopsis thaliana

Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear and mitochondrial encoded subunits for complex assembly in mitochondria await discovery. We generated knock-out lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis thaliana. Mutants had higher abundance of transcripts from mitochondrial genes encoding OXPHOS protein complexes, while transcripts for nuclear genes encoding other subunits of the same complexes showed no change in abundance. In contrast, the protein abundance of specific nuclear-encoded subunits in OXPHOS complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Protein complexes mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import, assembly and function of Complex I revealed that while function was retained, protein homeostasis was disrupted through decreased assembly, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes. One sentence summaryCLPP contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.

plant biology↗

bioRxiv2020

Advantages and limits of metagenomic assembly and binning of a giant virus

Giant viruses have large genomes, often within the size range of cellular organisms. This distinguishes them from most other viruses and demands additional effort for the successful recovery of their genomes from environmental sequence data. Here we tested the performance of genome-resolved metagenomics on a recently isolated giant virus, Fadolivirus, by spiking it into an environmental sample from which two other giant viruses were isolated. At high spike-in levels, metagenome assembly and binning led to the successful genomic recovery of Fadolivirus from the sample. A complementary survey of viral hallmark genes indicated the presence of other giant viruses in the sample matrix, but did not detect the two isolated from this sample. Our results indicate that genome-resolved metagenomics is a valid approach for the recovery of near-complete giant virus genomes given that sufficient clonal particles are present. Our data also underline that a vast majority of giant viruses remain currently undetected, even in an era of terabase-scale metagenomics.

microbiology↗

bioRxiv2019

Noncoding de novo mutations contribute to autism spectrum disorder via chromatin interactions

Three-dimensional chromatin structures regulate gene expression across genome. The significance of de novo mutations (DNMs) affecting chromatin interactions in autism spectrum disorder (ASD) remains poorly understood. We generated 931 whole-genome sequences for Korean simplex families to detect DNMs and identified target genes dysregulated by noncoding DNMs via long-range chromatin interactions between regulatory elements. Notably, noncoding DNMs that affect chromatin interactions exhibited transcriptional dysregulation implicated in ASD risks. Correspondingly, target genes were significantly involved in histone modification, prenatal brain development, and pregnancy. Both noncoding and coding DNMs collectively contributed to low IQ in ASD. Indeed, noncoding DNMs resulted in alterations, via chromatin interactions, in target gene expression in primitive neural stem cells derived from human induced pluripotent stem cells from an ASD subject. The emerging neurodevelopmental genes, not previously implicated in ASD, include CTNNA2, GRB10, IKZF1, PDE3B, and BACE1. Our results were reproducible in 517 probands from MSSNG cohort. This work demonstrates that noncoding DNMs contribute to ASD via chromatin interactions.

neuroscience↗

bioRxiv2019

Geometric regulation of histone state directs melanoma reprogramming

Malignant melanoma displays a high degree of cellular plasticity during disease progression, making classification of the heterogeneous population and selection of an appropriate therapy challenging. Signals in the tumor microenvironment are believed to influence melanoma plasticity through changes in the epigenetic state to guide dynamic differentiation and de-differentiation events that underlie tumorigenicity and dissemination. Here we uncover a relationship between geometric features at perimeter regions of multicellular melanoma aggregates, and reprogramming to a stem cell-like melanoma initiating cell (MIC) through histone marks H3K4Me2 and H3K9Ac. Using an in vitro tumor microengineering approach, we find concurrent expression of molecular MIC markers and spatial enrichment of these histone modifications at perimeter features. Chromatin immunoprecipitation and sequencing analysis demonstrates broad regulation of genes associated with SOX-, ETS-, and USF-families. SOX10 and PRDM14, transcriptional regulators with a putative role in several cancers, overlap with H3K9Ac and show elevated expression in cells along regions of perimeter curvature. siRNA knockdown of the epigenetic modifier PRDM14 abolishes the MIC phenotype suggesting a role in regulating melanoma heterogeneity. Our results suggest mechanotransduction at the periphery of melanoma tumors may orchestrate the activity of epigenetic modifiers to regulate histone state, cellular plasticity, and tumorigenicity.

cancer biology↗

bioRxiv2019

Fluorescence-based whole plant imaging and phenomics

Reverse genetics approaches have revolutionized plant biology and agriculture. Phenomics has the prospect of bridging plant phenotypes with genes, including transgenes, to transform agricultural fields1. Genetically-encoded fluorescent proteins (FPs) have transformed studies in gene expression, protein trafficking, and plant physiology. While the first instance of plant canopy imaging of green fluorescent protein (GFP) was performed over 20 years ago2, modern phenomics has largely ignored fluorescence as a transgene indicator despite the burgeoning FP color palette currently available to biologists3-5. Here we show a new platform for standoff imaging of plant canopies expressing a wide variety of FP genes in leaves. The platform, the fluorescence-inducing laser projector (FILP), uses a low-noise camera to image a scene illuminated by compact diode lasers of various colors and emission filters to phenotype transgenic plants expressing multiple constitutive or inducible FPs. Of the 20 FPs screened, we selected the top performing candidates for standoff phenomics at [≥] 3 m using FILP in a laboratory-based laser range. Included in demonstrated applications is the performance of an osmotic stress-inducible synthetic promoter selected from a high throughput library screen. While FILP has unprecedented versatility as a laboratory platform, we envisage future iterations of the system for use in automated greenhouse or even drone-fielded versions of the platform for crop screening.

plant biology↗

bioRxiv2019

Efficient Homology-directed Repair with Circular ssDNA Donors

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

molecular biology↗

bioRxiv2019

Genetically encoded phase contrast agents for digital holographic microscopy

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles, a unique class of air-filled protein nanostructures derived from buoyant microbes. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, greatly expanding the capabilities of this powerful technology for three-dimensional biological imaging.

bioengineering↗

bioRxiv2019

Phosphatidylinositol 4-kinase III beta regulates cell shape, migration and focal adhesion number.

Cell shape is regulated by cell adhesion and cytoskeletal and membrane dynamics. Cell shape, adhesion and motility have a complex relationship and understanding them is important in understanding developmental patterning and embryogenesis. Here we show that the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIII{beta}) regulates cell shape, migration and Focal Adhesion (FA) number. PI4KIII{beta} generates phosphatidylinositol 4-phosphate from phosphatidylinositol and is highly expressed in a subset of human breast cancers. PI4KIII{beta} and the PI4P it generates regulate a variety of cellular functions, ranging from control of Golgi structure, fly fertility and Akt signaling. Here, we show that loss of PI4KIII{beta} expression decreases cell migration and alters cell shape in NIH3T3 fibroblasts. The changes are accompanied by an increase in the number of FA in cells lacking PI4KIII{beta}. Furthermore, we find that PI4P-containing vesicles move to the migratory leading-edge during migration and that some of these vesicles tether to and fuse with FA. Fusion is associated with FA disassembly. This suggests a novel regulatory role for PI4KIII{beta} and PI4P in cell adhesion and cell shape maintenance.

cell biology↗

bioRxiv2019

Phosphorylation of the CAMTA3 transcription factor triggers its destabilization and nuclear export.

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes.\n\nOne sentence summaryTreatment with flg22 activates two independent kinase pathways that effect CAMTA3 phosphorylation and degradation.

plant biology↗

bioRxiv2019

High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

Phosphate overload contributes to mineral bone disorders associated with crystal nephropathies. Phytate, the major form of phosphorus in plant seeds, is known as an indigestible and negligible in humans. However, the mechanism and adverse effects of high-phytate intake on Ca2+ and phosphate absorption and homeostasis are unknown. Here we show that excessive intake of phytate with a low-Ca2+ diet fed to rats contributed to the development of crystal nephropathies, renal phosphate wasting, and bone loss through tubular dysfunction secondary to dysregulation of intestinal calcium and phosphate absorption. Moreover, Ca2+ supplementation alleviated the detrimental effects of excess dietary phytate on bone and kidney through excretion of undigested Ca2+-phytate, which prevented a vicious cycle of intestinal phosphate overload and renal phosphate wasting while improving intestinal Ca2+ bioavailability. Thus, we demonstrate that phytate is digestible without a high-Ca2+ diet and a risk factor for phosphate overloading and developing crystal nephropathies and bone disease.

physiology↗