Biology subjects

Cao, G.

Publications and source records attributed to Cao, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Genomic evolution reshapes cell type diversification in the amniote brain

Over 320 million years of evolution, amniotes have developed complex brains and cognition through largely unexplored genetic and gene expression mechanisms. We created a comprehensive single-cell atlas of over 1.3 million cells from the telencephalon and cerebellum of turtles, zebra finches, pigeons, mice, and macaques, employing single-cell resolution spatial transcriptomics to validate gene expression patterns across species. Our study revealed significant species-specific variations in cell types, highlighting their conservation and diversification in evolution. We found pronounced differences in telencephalon excitatory neurons (EX) and cerebellar cell types between birds and mammals. Birds predominantly express SLC17A6 in EX, whereas mammals expressed SLC17A7 in neocortex and SLC17A6 elsewhere, possibly due to loss of SLC17A7 function loss in birds. Additionally, we identified a novel bird-specific Purkinje cell subtype (SVIL+), implicating the LSD11/KDM1A pathway in learning and circadian rhythms, and related numerous positively selected genes in birds, suggesting an evolutionary optimization of cerebellar functions for ecological and behavioral adaptation. Our findings elucidate the complex interplay between genetic evolution and environmental adaptation, underscoring the role of genetic diversification in the development of specialized cell types across amniotes.

evolutionary biology↗

bioRxiv2024

Human striatal progenitor cells that contain inducible safeguards and overexpress BDNF rescue Huntington's disease phenotypes in R6/2 mice

Huntingtons disease (HD) is an autosomal-dominant neurodegenerative disorder characterized by striatal atrophy. Reduced trophic support due to decreased striatal levels of neurotrophins (NTs), mainly brain-derived neurotrophic factor (BDNF), contributes importantly to HD pathogenesis; restoring NTs has significant therapeutic potential. Human pluripotent stem cells (hPSC) offer a scalable platform for NT delivery but has potential safety risks including teratoma formation. We engineered hPSCs to constitutively produce BDNF and contain inducible safeguards to eliminate these cells if safety concerns arise. This study examined the efficacy of intrastriatally transplanted striatal progenitor cells (STRpcs) derived from these hPSCs against HD phenotypes in R6/2 mice. Engrafted STRpcs overexpressing BDNF alleviated motor and cognitive deficits and reduced mutant huntingtin aggregates. Activating the inducible safety switch with rapamycin safely eliminated the engrafted cells. These results demonstrate that BDNF delivery via a novel hPSC-based platform incorporating safety switches could be a safe and effective HD therapeutic.

neuroscience↗

bioRxiv2024

U-FISH: a universal deep learning approach for accurate FISH spot detection across diverse datasets

In the rapidly advancing landscape of fluorescence in situ hybridization (FISH) technologies, there is a critical need for sophisticated yet adaptable methods for spot detection. This study introduces U-FISH, a deep learning approach that significantly improves accuracy and generalization capabilities. Our method utilizes a U-Net model to transform noisy and ambiguous FISH images into a standardized representation with consistent signal characteristics, facilitating efficient spot detection. For the training and evaluation of the U-FISH model, we have constructed a comprehensive dataset comprising over 4,000 images and more than 1.6 million manually annotated spots, sourced from both experimental and simulated environments. Our benchmarks demonstrate that U-FISH outperforms existing methods for FISH spot detection, offering improved versatility by eliminating the need for laborious manual parameter adjustments. This allows for its application across a broad spectrum of datasets and formats. Furthermore, U-FISH is designed for high scalability and is capable of processing 3D data, supporting the latest generation of file formats for large and complex datasets. To promote community adoption and ensure accessibility, we provide a user-friendly interfaces: Napari plugin, web application and command-line interface. The complete training dataset is made publicly available, laying a solid foundation for future research in this field.

bioinformatics↗

bioRxiv2024

Role of histone variants H2BC1 and H2AZ.2 in H2AK119ub nucleosome organization and Polycomb gene silencing

Ubiquitination of histone H2A at lysine 119 residue (H2AK119ub) plays critical roles in a wide range of physiological processes, including Polycomb gene silencing 1,2, replication 3-5, DNA damage repair 6-10, X inactivation 11,12, and heterochromatin organization 13,14. However, the underlying mechanism and structural basis of H2AK119ub remains largely elusive. In this study, we report that H2AK119ub nucleosomes have a unique composition, containing histone variants H2BC1 and H2AZ.2, and importantly, this composition is required for H2AK119ub and Polycomb gene silencing. Using the UAB domain of RSF1, we purified H2AK119ub nucleosomes to a sufficient amount and purity. Mass spectrometry analyses revealed that H2AK119ub nucleosomes contain the histone variants H2BC1 and H2AZ.2. A cryo-EM study resolved the structure of native H2AK119ub nucleosomes to a 2.6A resolution, confirming H2BC1 in one subgroup of H2AK119ub nucleosomes. Tandem GST-UAB pulldown, Flag-H2AZ.2, and HA-H2BC1 immunoprecipitation revealed that H2AK119ub nucleosomes could be separated into distinct subgroups, suggesting their composition heterogeneity and potential dynamic organization. Knockout or knockdown of H2BC1 or H2AZ.2 reduced cellular H2AK119ub levels, establishing H2BC1 and H2AZ.2 as critical determinants of H2AK119ub. Furthermore, genomic binding profiles of H2BC1 and H2AZ.2 overlapped significantly with H2AK119ub binding, with the most significant overlapping in the gene body and intergenic regions. Finally, assays in developing embryos reveal an interaction of H2AZ.2, H2BC1, and RING1A in vivo. Thus, this study revealed, for the first time, that the H2AK119ub nucleosome has a unique composition, and this composition is required for H2AK119ub and Polycomb gene silencing.

molecular biology↗

bioRxiv2024

Cardiac stress leads to regulation of Filamin C dimerisation via an ancient phosphorylation-modulated interaction with HSPB7

The biomechanical properties and responses of tissues underpin a variety of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. Seemingly a multi-functional protein that interacts with a variety of partners, how FLNC is regulated at the molecular level is not well understood. Here we have investigated its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We found that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we have solved by means of X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work rationalises on the molecular level how FLNC might switch between stabilising functions in the cell, and reveals how HSPB7 acts as a specific molecular chaperone that regulates FLNC.

molecular biology↗

bioRxiv2023

Multi-task machine learning reveals the neuroanatomy fingerprint of mental processing

Mental processing delineates the functions of human mind encompassing a wide range of motor, sensory, emotional, and cognitive processes, each of which is underlain by the neuroanatomical substrates. Identifying accurate representation of functional neuroanatomy substrates of mental processing could inform understanding of its neural mechanism. The challenge is that it is unclear whether a specific mental process possesses a functional neuroanatomy fingerprint, i.e., a unique and reliable pattern of functional neuroanatomy that underlies the mental process. To address this question, we utilized a multi-task deep learning model to disentangle the functional neuroanatomy fingerprint of seven different and representative mental processes including Emotion, Gambling, Language, Motor, Relational, Social, and Working Memory. Results based on the functional magnetic resonance imaging data of two independent cohorts of 1235 subjects from the US and China consistently show that each of the seven mental processes possessed a functional neuroanatomy fingerprint, which is represented by a unique set of functional activity weights of whole-brain regions characterizing the degree of each region involved in the mental process. The functional neuroanatomy fingerprint of a specific mental process exhibits high discrimination ability (93% classification accuracy and AUC of 0.99) with those of the other mental processes, and is robust across different datasets and using different brain atlases. This study provides a solid functional neuroanatomy foundation for investigating the neural mechanism of mental processing. One-Sentence SummaryThere exists a functional neuroanatomy fingerprint to underlie the mental process.

bioinformatics↗

bioRxiv2023

Species -Shared and -Unique Gyral Peaks on Human and Macaque Brains

Cortical folding is an important feature of primate brains that plays a crucial role in various cognitive and behavioral processes. Extensive research has revealed both similarities and differences in folding morphology and brain function among primates including macaque and human. The folding morphology is the basis of brain function, making cross-species studies on folding morphology important for understanding brain function and species evolution. However, prior studies on cross-species folding morphology mainly focused on partial regions of the cortex instead of the entire brain. Previously, we defined a whole-brain landmark based on folding morphology: the gyral peak. It was found to exist stably across individuals and ages in both human and macaque brains. In this study, we identified shared and unique gyral peaks in human and macaque, and investigated the similarities and differences in the spatial distribution, anatomical morphology, and functional connectivity of them.

neuroscience↗

bioRxiv2023

Insights from the Early Cretaceous: The promise of Lycoptera aDNA sequencing

Obtaining high-quality ancient DNA (aDNA) is a crucial process for advancements in molecular paleontology. However, current research is restricted to a limited range of fossil types due to DNA degradation and contamination from environmental DNA (eDNA). Utilizing the technology of nanoparticle affinity beads, we successfully extracted DNA from Lycoptera davidi fossils and obtained 1,258,901 DNA sequences. To ascertain their origins, we developed and implemented a novel protocol, the "mega screen method", designed to categorize aDNA fragments. This approach successfully identified 243 Actinopterygii species-specific fragments (SSFs), which subsequent analysis confirmed as original in situ DNA (oriDNA) sequences. Lycoptera davidi, a ray-finned fish from the Early Cretaceous Jehol Biota, dates back approximately 120 million years (Ma). Notably, the absence of deamination in certain oriDNA and paleoenvironmental DNA (paeDNA) fragments calls into question its reliability as a definitive marker for aDNA identification, a phenomenon potentially attributable to the protective effects of fossil diagenesis. Additionally, we identified a novel mechanism, "coding region sliding replication and recombination", responsible for generating new transposase. This mechanism, distinct from conventional processes such as horizontal gene transfer or vertical inheritance from parents, can facilitate a rapid genomic diversification. The transposase CDS insertion into the GAS2 gene of Anabarilius grahami offers molecular insights into the genetic interactions between ancestral carp and Lycoptera and the swift emergence of the expansion of early fish species starting from the early Cretaceous. These findings enhance our understanding of fossil DNA preservation, lay the groundwork for an ancient species DNA database, and introduce innovative tools for exploring evolutionary relationships across geological timescales.

paleontology↗

bioRxiv2023

Morphine Re-arranges Chromatin Spatial Architecture of Primate Cortical Neurons

The expression of linear DNA sequences is precisely regulated by the three-dimensional (3D) architecture of chromatin. Morphine-induced aberrant gene networks of neurons have been extensively investigated; however, how morphine impacts the 3D genomic architecture of neuorns is still unknown. Here, we applied digestion-ligation-only high-throughput chromosome conformation capture (DLO Hi-C) technology to investigate the affection of morphine on 3D chromatin architecture of primate cortical neurons. After receiving continuous morphine administration for 90 days on rhesus monkeys, we discovered that morphine re-arranged chromosome territories, with a total of 391 segmented compartments being switched. Morphine altered over half of the detected topologically associated domains (TADs), most of which exhibited a variety of shifts, followed by separating and fusing types. Analysis of the looping events at kilobase-scale resolution revealed that morphine increased not only the number but also the length of differential loops. Moreover, all identified differentially expressed genes (DEGs) from the RNA sequencing (RNA-seq) were mapped to the specific TAD boundaries or differential loops, and were further validated to be significantly changed. Collectively, an altered 3D genomic architecture of cortical neurons may regulate the gene networks associated-morphine effects. Our finding provides critical hubs connecting chromosome spatial organization and gene networks associated with the morphine effects in humans.

bioinformatics↗

bioRxiv2023

Development of Multiomics in situ Pairwise Sequencing (MiP-Seq) for Single-cell Resolution Multidimensional Spatial Omics

Delineating the spatial multiomics landscape will pave the way to understanding the molecular basis of physiology and pathology. However, current spatial omics technology development is still in its infancy. Here, we developed a high-throughput multiomics in situ pairwise sequencing (MiP-Seq) strategy to efficiently decipher multiplexed DNAs, RNAs, proteins, and small biomolecules at subcellular resolution. We delineated dynamic spatial gene profiles in the hypothalamus using MiP-Seq. Moreover, MiP-Seq was unitized to detect tumor gene mutations and allele-specific expression of parental genes and to differentiate sites with and without the m6A RNA modification at specific sites. MiP-Seq was combined with in vivo Ca2+ imaging and Raman imaging to obtain a spatial multiomics atlas correlated to neuronal activity and cellular biochemical fingerprints. Importantly, we proposed a "signal dilution strategy" to resolve the crowded signals that challenge the applicability of in situ sequencing. Together, our method improves spatial multiomics and precision diagnostics and facilitates analyses of cell function in connection with gene profiles.

molecular biology↗

bioRxiv2022

Robust identification of extrachromosomal DNA and genetic variants using multiple genetic abnormality sequencing (MGA-Seq)

Genomic abnormalities, including structural variation (SV), copy number variation (CNV), single-nucleotide polymorphism (SNP), homogenously staining regions (HSR) and extrachromosomal DNA (ecDNA), are strongly associated with cancer, rare diseases and infertility. A robust technology to simultaneously detect these genomic abnormalities is highly desired for clinical diagnosis and basic research. In this study, we developed a simple and cost-effective method - multiple genetic abnormality sequencing (MGA-Seq) - to simultaneously detect SNPs, CNVs, SVs, ecDNA and HSRs in a single tube. This method has been successfully applied in both cancer cell lines and clinical tumour samples and revealed that focal amplification in tumour tissue is substantially heterogeneous. Notably, we delineated the architecture of focal amplification and the ecDNA network by MGA-Seq, which facilitated the exploration of the regulation of gene expression in ecDNA. This method could be extensively applied for diagnosis and may greatly facilitate the investigation of the genomic mechanism for genetic diseases.

genomics↗

bioRxiv2022

Phosphatidylethanolamine facilitates mitochondrial pyruvate entry to regulate metabolic flexibility

Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation (OXPHOS). Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness by small mouse cage (SMC) that, unlike other classic models of disuse in mice, faithfully recapitulates metabolic responses that occur in humans. Bioenergetic phenotyping of mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE was sufficient to increase glucose flux towards lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a novel mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility.

physiology↗

bioRxiv2022

Machine learning enables accurate prediction of breast cancer five-year survival using somatic genomic variants

Breast cancer is one of the most common cancers, accounting for about 30% of female cancers and a mortality rate of 15%. The 5-year survival rate is most commonly used to assess cancer progression and guide clinical practice. We used the CatBoost model to systematically construct a five-year mortality risk prediction model based on two independent data sets (BRCA_METABRIC, BRCA_TCGA). The model input data are the somatic genomic variants (copy number variation, SNP locus, cumulative mutation number of genes) and phenotype data of cancer samples. The optimal model combined all the above characteristics, and the AUC reached 0.70 in an independent external data set. At the same time, we also conducted a biological analysis of the characteristics of the model and found some potential biomarkers (TP53, DNAH11, MAP3K1, PHF20L1, etc.). The results of model risk stratification can be used as a guide for the prognosis of breast cancer.

bioinformatics↗

bioRxiv2022

Isometric Spiracular Scaling in Scarab Beetles: Implications for Diffusive and Advective Oxygen Transport

The scaling of respiratory structures has been hypothesized to be a major driving factor in the evolution of many aspects of animal physiology. Here we provide the first assessment of the scaling of the spiracles in insects using ten scarab beetle species differing 180x in mass, including some of the most massive extant insect species. Using X-ray microtomography, we measured the cross-sectional area and depth of all eight spiracles, enabling the calculation of their diffusive and advective capacities. Each of these metrics scaled with geometric isometry.Because diffusive capacities scale with lower slopes than metabolic rates, the largest beetles measured require 10-fold higher PO2 gradients across the spiracles to sustain metabolism by diffusion compared to the smallest species. Large beetles can exchange sufficient oxygen for resting metabolism by diffusion across the spiracles, but not during flight. In contrast, spiracular advective capacities scale similarly or more steeply than metabolic rates, so spiracular advective capacities should match or exceed respiratory demands in the largest beetles. These data illustrate a general principle of gas exchange: scaling of respiratory transport structures with geometric isometry diminishes the potential for diffusive gas exchange but enhances advective capacities; combining such structural scaling with muscle-driven ventilation allows larger animals to achieve high metabolic rates when active.

physiology↗

bioRxiv2022

Microbial dysbiosis and metabolic disorders promote rheumatoid arthritis progression across successive stages

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease that typically progresses through four stages, from mild stiffness to severe disability. The critical involvement of gut microbial dysbiosis in promoting RA has been intriguing. The aim of this study was to investigate stage-specific roles of microbial dysbiosis and metabolic disorders in pathogenesis across successive stages of RA. MethodsIn this multi-omics cohort study, fecal metagenomic, plasma metabolomic data were collected from 76 patients grouped into four RA stages, 19 patients with osteoarthritis, and 27 healthy individuals in China. A non-redundant gene catalogue was constructed, functionally annotated, and clustered into metagenomic species (MGS). Leave-one-out analysis was performed to determine the MGS that most drove the microbial dysfunctions with RA across stages. Random forest algorithm was performed to investigate diagnostic potential of microbial or metabolic features for each stage. Additionally, to verify the bacterial invasion of the joint synovial fluid, we conducted 16S rRNA gene sequencing, bacterial isolation, and scanning electron microscopy on joint synovial fluid from another cohort of 271 RA patients grouped into four RA stages. FindingsWe found that microbial dysbiosis and metabolic disorders had stage-specific effects on RA progression. For instance, arginine succinyltransferase pathway was elevated mainly in the second and third stages of RA (p=1{middle dot}4x10-3), which was driven by Escherichia coli, and it was correlated with the increase of the rheumatoid factor (p=1{middle dot}35x10-3). Considerable elevation of methoxyacetic acid (p=1{middle dot}28x10-8) and cysteine-S-sulfate (p=4{middle dot}66x10-12) might inhibit osteoblasts in the second stage and enhance osteoclasts in the third stage, respectively. Moreover, fecal microbes Eggerthella lenta and Bifidobacterium longum that were exclusively elevated in the fourth stage, were also detected in the joint synovial fluid. InterpretationOur findings elucidate for the first time the stage-specific roles of microbial dysbiosis and metabolic disorders across successive stages of RA, which open up new avenues for RA prognosis and therapy. We demonstrate the buildup of these effects might induce microbial invasion of the joint synovial fluid in the fourth stage of RA.

microbiology↗

bioRxiv2022

LPS-TLR4 pathway stimulating in acute-on-chronic liver failure by promote NETs formation

Backgrounds: Intrahepatic infiltration of neutrophils is a character of alcoholic acute-on-chronic liver failure (AACLF) and neutrophil extracellular traps (NETs) are an important strategy for neutrophils to fix and kill invading microorganisms. Intestinal bacteria and the gut-liver axis have been thought to play a key role in many liver diseases also including AACLF. However, whether NETs appear in AACLF and play a role in AACLF is still unsure. Methods: WT, NE KO, and TLR4 KO mice were used to build the AACLF model, and the intestinal bacteria were eliminated at the same time and LPS was given. Then the formation of NETs and AACLF related markers were detected. Results: The serum MPO-DNA and LPS concentration was increased in AACLF patients and a correlation was revealed between these two indexes. More intrahepatic NETs formed in AACLF mice by testing MPO-DNA, Cit H3, and NE. These markers decreased with gut detergent and restored markers with gut detergent plus LPS supplement. While NETs formation failed to change with gut microbiome or combine LPS supplement in TLR4 KO mice. As we tested AACLF related characters, liver injury, intrahepatic fat deposition, inflammation, and fibrosis alleviated with depletion of NE. These related marks were also attenuated with gut sterilization by antibiotics and recovered with combined treatment with antibiotics plus LPS. But the liver injury, intrahepatic fat, fibro deposition, and liver inflammation-related markers did show a significant difference in TLR4 KO mice when they received the same treatment. Conclusion: Intestinal-derived LPS promotes NETs formation in AACLF through the TLR4 pathway and further accelerates the AACLF process by NETs.

pathology↗

bioRxiv2022

A single-cell atlas reveals shared and distinct immune responses and metabolism during SARS-CoV-2 and HIV-1 infections

SARS-CoV-2 and HIV-1 are RNA viruses that have killed millions of people worldwide. Understanding the similarities and differences between these two infections is critical for understanding disease progression and for developing effective vaccines and therapies, particularly for 38 million HIV-1+ individuals who are vulnerable to SARS-CoV-2 co-infection. Here, we utilized single-cell transcriptomics to perform a systematic comparison of 94,442 PBMCs from 7 COVID-19 and 9 HIV-1+ patients in an integrated immune atlas, in which 27 different cell types were identified using an accurate consensus single-cell annotation method. While immune cells in both cohorts show shared inflammation and disrupted mitochondrial function, COVID-19 patients exhibit stronger humoral immunity, broader IFN-I signaling, elevated Rho GTPase and mTOR pathway activities, and downregulated mitophagy. Our results elucidate transcriptional signatures associated with COVID-19 and HIV-1 that may reveal insights into fundamental disease biology and potential therapeutic targets to treat these viral infections. HighlightsO_LICOVID-19 and HIV-1+ patients show disease-specific inflammatory immune signatures C_LIO_LICOVID-19 patients show more productive humoral responses than HIV-1+ patients C_LIO_LISARS-CoV-2 elicits more enriched IFN-I signaling relative to HIV-I C_LIO_LIDivergent, impaired metabolic programs distinguish SARS-CoV-2 and HIV-1 infections C_LI

bioinformatics↗

bioRxiv2021

Modularity and selection of nectar traits in the evolution of the selfing syndrome in Ipomoea lacunosa (Convolvulaceae)

O_LIAlthough the evolution of the selfing syndrome often involves reductions in floral size, pollen, and nectar, few studies of selfing syndrome divergence have examined nectar. We investigate whether nectar traits have evolved independently of other floral size traits in the selfing syndrome, whether nectar traits diverged due to drift or selection, and the extent to which quantitative trait locus (QTL) analyses predict genetic correlations. C_LIO_LIWe use F5 recombinant inbred lines (RILs) generated from a cross between Ipomoea cordatotriloba and I. lacunosa. We calculate genetic correlations to identify evolutionary modules, test whether traits have been under selection, identify QTLs, and perform correlation analyses to evaluate how well QTL properties reflect the genetic correlations. C_LIO_LINectar and floral size traits form separate genetic clusters. Directional selection has acted to reduce nectar traits in the selfing I. lacunosa. Calculations from QTL properties are consistent with observed genetic correlations. C_LIO_LIFloral trait divergence during mating system syndrome evolution reflects independent evolution of at least two evolutionary modules: nectar and floral size traits. This independence implies that adaptive change in these modules requires direct selection on both floral size and nectar traits. Our study also supports the expected mechanistic link between QTL properties and genetic correlations. C_LI

evolutionary biology↗