Biology subjects

Xu, X.

Publications and source records attributed to Xu, X..

At least 649 records · Page 36Linked to original sources

bioRxiv2020

Single cell atlas of trisomy 21 cerebral cortex

Down syndrome (DS) is one of the most common human birth defects caused by trisomy 21 (T21), leading to a variety of cognitive impairments. The cellular composition of human brain has been explored using single cell RNA sequencing in both physiological and pathological conditions. However, the cellular heterogeneity of human brain with chromosome aneuploidy is largely unknown. Here, we profiled the transcriptome of 36046 cells in cerebral cortex of T21 human fetus, covering frontal lobe, parietal lobe, occipital lobe and temporal lobe. Intriguingly, we detected several genes positively associated with neurons maturation was dysregulated in T21 frontal cortex (HIC2, POU2F2, ZGLP1 and FOXK1). To share, explore and utilized the data resources of T21 cerebral cortex, we developed a comprehensive platform named T21atlas, composing of two functional modules (T21cluster and T21talk). Overall, our study provides, as far as we know, the first single cell atlas for T21 cerebral cortex, which could promote our understanding of the molecular mechanism of DS at an unprecedented resolution and could potentially facilitate the development of novel clinical therapeutics against T21.

neuroscience↗

bioRxiv2019

Acetylation is required for NLRP3 self-aggregation and full activation of the inflammasome

The full activation of NLRP3 inflammasome needs two sequential signals: the fist priming signal and the second assembly signal. Various stimuli including infections and stress signals can provide the assembly signal. However, how NLRP3 detects diverse stimuli and becomes fully activated remain largely unknown. In this study, we found the second signal specially triggers the acetylation of NLRP3, which facilitates the aggregation of NLRP3 and its interaction with ASC and NEK7, thus promoting the assembly of inflammasome. Meanwhile, by employing pharmacological and molecular approaches, we identified KAT5 as a regulator of NLRP3 acetylation and activation. Furthermore, KAT5 specific inhibitor-NU9056 exhibited a robust suppressive effect on NLRP3 inflammasome both in vitro and in vivo. Thus, our study reveals a new mechanism for NLRP3 full activation and suggests targeting NLRP3 acetylation may provide a new approach for treatment of NLRP3 associated diseases.

immunology↗

bioRxiv2019

Advanced Whole Genome Sequencing Using a Complete PCR-free Massively Parallel Sequencing (MPS) Workflow

BackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling. ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope). ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

genomics↗

bioRxiv2019

NADPH diaphorase neuronal dystrophy in gracile nucleus, cuneatus nucleus and spinal trigeminal nucleus in aged rat

NADPH-diaphorase (N-d) activity is commonly used to identify NOS-ergic neurons. In our previous study, N-d positive neuritic dystrophy and spheroid termed aging-related N-d Body is discovered in the lumbosacral spinal cord in the normal aging rats. Histological studies also reveal that N-d positive neurodegenerative changes occur in the gracile nucleus. We re-examined N-d activity in gracile nucleus in aged rat. We found N-d positive neuritic dystrophy and spheroid also occurred in the cuneatus nucleus and spinal trigeminal nucleus. Besides regular coronal section, longitudinal oriented dystrophic neurites were detected in the sagittal and horizontal section in gracile nucleus and dorsal column. We fziurther examined the medullary oblongata with regular classical histology including Golgi staining, immunocytochemistry of NOS and phosphorylated tau protein, neuronal tracing method with wheat germ agglutinin conjugated alexa-fluor-488 through sciatic nerve, and spinal cord transection at thoracic level. Most of N-d positive neuritic dystrophy and spheroid did not showed colocalization with NOS or phosphorylated tau protein. Neuronal tracing and spinal cord transection revealed that N-d dystrophic neurites in gracile nucleus originated from terminal of sensory projection from spinal cord and peripheral somatic input. The results suggested that aging-related N-d dystrophy in the gracile nucleus was unique morphological feature. In conclusion, it was postulated that the N-d dystrophy as a morphological marker of aging degenerative damage in normal aged organisms.

neuroscience↗

bioRxiv2019

mtor Haploinsufficiency Ameliorates Renal Cyst Formation in Adult Zebrafish tmem67 Mutants

Although zebrafish embryos have been utilized to study ciliogenesis and to model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Here, we report the generation and characterization of a zebrafish mutant of tmem67, a homologue of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Although a small population of homozygous embryos exhibited pronephric cysts, all mutants were able to survive to adulthood and developed progressive mesonephric cysts with full penetrance. The cysts in the adult zebrafish kidneys manifested features of mammalian PKD, including switching of cyst origin from the proximal tubules to the collecting ducts, increased proliferation of cyst-lining epithelial cells, and hyperactive mTOR signaling. Consistent ciliary abnormalities were observed in both the embryonic and adult zebrafish mutants compared with the wild-type fish, including shorter and fewer single cilia in the distal pronephros and all segments of the mesonephros and greater numbers of multiciliated cells (MCCs). Lack of single cilium preceded cystogenesis, suggestive of a primary defect. Expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of cystogenesis in young adult zebrafish, suggesting an adaptive action. Interestingly, mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. In summary, we have established a tmem67 mutant as the first adult zebrafish PKD model, revealed a novel aspect of cilium regulation, and identified sustained mTOR inhibition as a candidate therapeutic strategy for tmem67-based PKD. Significance StatementWhile zebrafish embryos are well recognized for their value in studying ciliogenesis and polycystic kidney disease (PKD), adult zebrafish have not commonly been used. Here, we report the establishment of the first adult zebrafish model for PKD, which exhibits characteristics of mammalian PKD and shows kidney ciliary abnormalities consistent with those observed in an embryonic model. We also provide evidence for mTOR inhibition as a therapeutic strategy for this particular type of cystogenesis. Compared to the embryonic model, the adult fish model exhibits a spectrum of progressive pathogeneses and enables ciliary abnormalities to be discerned as either primary or secondary to cystogenesis. We believe that this novel adult fish model will facilitate mechanistic studies and therapeutic development for PKD.

genetics↗

bioRxiv2019

Effects of Endophytic Fungus MF23 on Dendrobium nobile Lindl. in an artificial primary environment

The quality of Dendrobium nobile Lindl. is related to the endophytic fungus. It had reported that the mycorrhizal fungus MF23 helps to increase the content of dendrobine, but few studies had explained the mechanism of the phenomenon. In previous study, we verified the symbiotic mechanism of the mycorrhizal fungus MF23 with D. nobile on the agar medium. In this study, the research carried on the bark medium, nearly like the natural environment, which had special meaning because of its benefits for the widely application. As a result, it showed a significant effect especially in the later period of the cultivation, suggesting that mycorrhizal fungus MF23 had a promotion for D. nobile in the natural environment, which enabled the application of the technique in the experimental field. It also implied that post-modification enzyme genes might play an important role in stimulating biosynthesis of dendrobine.

genetics↗

bioRxiv2019

Single cell atlas of domestic pig brain illuminates the conservation and divergence of cell types at spatial and species levels

Domestic pig (Sus scrofa domesticus) has drawn much attention from researchers worldwide due to its implications in evolutionary biology, regenerative medicine and agriculture. The brain atlas of Homo sapiens (primate), Mus musculus (rodent), Danio rerio (fish) and Drosophila melanogaster (insect) have been constructed at single cell resolution, however, the cellular compositions of pig brain remain largely unexplored. In this study, we investigated the single-cell transcriptomic profiles of five distinct regions of domestic pig brain, from which we identified 21 clusters corresponding to six major cell types, characterized by unique spectrum of gene expression. By spatial comparison, we identified cell types enriched or depleted in certain brain regions. Inter-species comparison revealed cell-type similarities and divergences in hypothalamus between mouse and pig, providing invaluable resources for the evolutionary exploration of brain functions at single cell level. Besides, our study revealed cell types and molecular pathways closely associated with several diseases (obesity, anorexia, bulimia, epilepsy, intellectual disability, and autism spectrum disorder), bridging the gap between gene mutations and pathological phenotypes, which might be of great use to the development precise therapies against neural system disorders. Taken together, we reported, so far as we know, the first single cell brain atlas of Sus scrofa domesticus, followed by comprehensive comparisons across brain region and species, which could throw light upon future evo-devo, regenerative medicine, and agricultural studies.

bioinformatics↗

bioRxiv2019

Draft Genomes of two Artocarpus plants, Jackfruit (A. heterophyllus) and Breadfruit (A. altilis)

Two of the most economically important plants in the Artocarpus genus are jackfruit (A. heterophyllus Lam.) and breadfruit (A. altilis (Parkinson) Fosberg). Both species are long-lived trees that have been cultivated for thousands of years in their native regions. Today they are grown throughout tropical to subtropical areas as an important source of starch and other valuable nutrients. There are hundreds of breadfruit varieties that are native to Oceania, of which the most commonly distributed types are seedless triploids. Jackfruit is likely native to the western Ghats of India and produces one of the largest tree-borne fruit structures (reaching up to 100 pounds). To date, there is limited genomic information for these two economically important species. Here, we generated 273 Gb and 227 Gb of raw data from jackfruit and breadfruit, respectively. The high-quality reads from jackfruit were assembled into 162,440 scaffolds totaling 982 Mb with 35,858 genes. Similarly, the breadfruit reads were assembled into 180,971 scaffolds totaling 833 Mb with 34,010 genes. A total of 2,822 and 2,034 expanded gene families were found in jackfruit and breadfruit, respectively, enriched in pathways including starch- and sucrose metabolism, photosynthesis and others. The copy number of several starch synthesis related genes were found increased in jackfruit and breadfruit compared to closely related species, and the tissue specific expression might imply their sugar-rich and starch-rich characteristics. Overall, the publication of high-quality genomes for jackfruit and breadfruit provides information about their specific composition and the underlying genes involved in sugar and starch metabolism.

genomics↗

bioRxiv2019

A Novel LysR-Type Global Regulator RpvA Controls Persister Formation and Virulence in Staphylococcus aureus

Staphylococcus aureus is the leading cause of wound and nosocomial infections. Persister formation and virulence factors play crucial roles during S. aureus infection. However, the mechanisms of persister formation and its relationship to virulence in S. aureus are poorly understood. In this study, we screened a transposon mutant library and identified a LysR-type global transcriptional regulator NWMN_0037, which we called RpvA, for regulator of persistence and virulence, whose mutation leads to higher susceptibility to antibiotics ampicillin and norfloxacin and various stresses including oxidative stress, heat, and starvation in late exponential and early stationary phase. Interestingly, the rpvA mutant was highly attenuated for virulence compared with the parent S. aureus Newman strain as shown by a much higher lethal dose, reduced ability to survive in macrophages and to form abscess in the mouse model. Transcriptional profiling and metabolomic analysis revealed that RpvA could repress multiple genes including gapR, gapA, tpi, pgm, eno, glpD, and acs expression and enhance production of numerous intermediate metabolites including dihydroxyacetone phosphate, 2-phosphoglycerate, acetyl-CoA, glycerol 3-phosphate, L-glutamate in the cells. The differentially expressed genes and altered production of metabolites are distributed in global metabolism including carbohydrate metabolism, amino acid metabolism, energy metabolism and metabolism of cofactors and vitamins. These metabolic adjustments could cause the cell to go into dormancy, thus promoting S. aureus to convert to persisters. In addition, RpvA could upregulate the expression of virulence genes including hla, hlgA, hlgB, hlgC, lukF, lukS, lukD, sea and coa, and carotenoid biosynthesis genes (crtI, crtM, crtN). Gel shift assay confirmed that RpvA could bind to the promoters of candidate target genes hla, hlgB and crtM, thus promoting S. aureus virulence. Because of the important functions of the RpvA, it may serve as an attractive target for developing new drugs and vaccines to more effectively control S. aureus infections.

microbiology↗

bioRxiv2019

Porous upconversion nanostructures as bimodal biomedical imaging contrast agents

Lanthanide ions doped upconversion nanoparticles (UCNPs) hold great promise as the imaging contrast agent for multimodal medical imaging techniques for diagnosis, including fluorescent bioimaging, magnetic resonance imaging, and computed tomography. However, the maximized signal values of fluorescence and MRI cannot be achieved simultaneously from the same upconversion nanoparticles structures because high specific surface areas can benefit the signal gaining of MRI while big size can induce brighter fluorescent imaging. In this work, we designed and fabricated novel core-porous shell structures for UCNPs with much-enhanced signal values for both fluorescent imaging and MRI. The core-porous shell UCNPs were synthesized via a post-treatment process after an inert shell was coated onto the core UCNPs. The formation mechanism was carefully investigated. The fluorescent and magnetic resonance properties have been detailed characterized and compared from core, core-shell and core-porous UCNPs. Large and bright UCNPs in fluorescence and MRI have been achieved and great potential as the dual-modal contrast agent.

biophysics↗

bioRxiv2019

Slicing the genome of star-fruit (Averrhoa carambola L.)

The Averrhoa carambola is commonly known as star fruit because of its peculiar shape and its fruit is a rich source of minerals and vitamins. It is also used in traditional medicines in countries like India, China, the Philippines, and Brazil for treating various ailments such as fever, diarrhea, vomiting, and skin disease. Here we present the first draft genome of the Oxalidaceae family with an assembled genome size of 470.51 Mb. In total, 24,726 protein-coding genes were identified and 16,490 genes were annotated using various well-known databases. The phylogenomic analysis confirmed the evolutionary position of the Oxalidaceae family. Based on the gene functional annotations, we also discovered the enzymes possibly involved in the important nutritional pathways in star fruit genome. Overall, being the first sequenced genome in the Oxalidaceae family, the data provides an essential resource for the nutritional, medicinal, and cultivational studies for this economically important star-fruit plant.

genomics↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2019

Towards Practical and Robust DNA-based Data Archiving by Codec System Named ‘Yin-Yang’

DNA is a promising data storage medium due to its remarkable durability and space-efficient storage. Early bit-to-base transcoding schemes have primarily pursued information density, at the expense however of introducing biocompatibility challenges or at the risk of decoding failure. Here, we propose a robust transcoding algorithm named the "Yin-Yang Codec" (YYC), using two rules to encode two binary bits into one nucleotide, to generate DNA sequences highly compatible with synthesis and sequencing technologies. We encoded two representative file formats and stored them in vitro as 200-nt oligo pools and in vivo as an ~54-kb DNA fragment in yeast cells. Sequencing results show that YYC exhibits high robustness and reliability for a wide variety of data types, with an average recovery rate of 99.94% at 104 molecule copies and an achieved recovery rate of 87.53% at 100 copies. In addition, the in vivo storage demonstration achieved for the first time an experimentally measured physical information density of 198.8 EB per gram of DNA (44% of the theoretical maximum for DNA).

synthetic biology↗

bioRxiv2019

TGS-GapCloser: fast and accurately passing through the Bermuda in large genome using error-prone third-generation long reads

The completeness and accuracy of genome assemblies determine the quality of subsequent bioinformatics analysis. Despite benefiting from the medium/long-range information of third-generation sequencing techniques, current gap-closing tools to enhance assemblies suffer multi-alignments and high error rates, resulting in huge time and money costs.\n\nWe developed a software tool, TGS-GapCloser that uses the low depth (>=10X) single molecule sequencing long reads without any error correction to close gaps. The algorithm distinguishes gap regions from the alignments of long reads against original scaffolds, corrects only the candidate regions, and assigns the best sequences to each gap. We demonstrate that TGS-GapCloser improves the contig N50 value of draft assembly by 25-fold on average, updating above 90% gaps with 93.96% positive predictive value. Despite of high error rate of raw long reads, improved assemblies archive Q50 (99.999%) single-base accuracy with only 11.8% decrement to inputs. Besides it could complete more gaps, and is also [~]29-fold faster than mainstream gap-closing tools. BUSCO analysis revealed that 3.4%-13.1% more expected genes were complete. TGS-GapCloser also shows its power to fill gaps for ultra large genome assembly of ginkgo ([~]12Gb) with 71.6% of gaps closed. The validation of inserted or merged gap sequences was conducted with NGS reads and reference genomes, respectively. The updated genome assemblies may promote the gene annotation, structure variant calling and thus improving the downstream analysis of ontogeny, phylogeny, and evolution.

bioinformatics↗

bioRxiv2019

DNB-Based On-Chip Motif Finding (DocMF): a High-Throughput Method to Profile Different Types of Protein-DNA Interactions

Here we report a highly sensitive DNB-based on-chip Motif Finding (DocMF) system that utilizes high throughput next-generation-sequencing (NGS) chips to profile protein binding or cleaving activity. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer-adjacent-motif (PAM) sequences of different CRISPR systems. Our DocMF platform can simultaneously screen both 5 and 3 PAM regions with high coverage using the same NGS library/chip. For the well-studied SpCas9, our DocMF platform identified a small proportion of noncanonical 5-NAG-3 ([~]5%) and 5-NGA-3 ([~]1.6%), in addition to its common PAMs, 5-NGG-3 ([~]89.9%). We also used the DocMF to assay two uncharacterized Cas endonucleases, VeCas9 and BvCpf1. VeCas9 PAMs were not detected by the conventional PAM depletion method. However, DocMF discovered that both VeCas9 and BvCpf1 required broader and more complicated PAM sequences for target recognition. VeCas9 preferred the R-rich motifs, whereas BvCpf1 used the T-rich PAMs. Moreover, after slightly changing the experimental protocol, we observed that dCas9, a DNA-binding protein lacking endonuclease activity, preferably binded to the previously reported PAMs 5-NGG-3. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA-binding proteins.

molecular biology↗

bioRxiv2019

Over 50000 metagenomically assembled draft genomes for the human oral microbiome reveal new taxa

The oral cavity of each person is home for hundreds of bacterial species. While taxa for oral diseases have been well studied using culture-based as well as amplicon sequencing methods, metagenomic and genomic information remain scarce compared to the fecal microbiome. Here we provide metagenomic shotgun data for 3346 oral metagenomics samples, and together with 808 published samples, assemble 56,213 metagenome-assembled genomes (MAGs). 64% of the 3,589 species-level genome bins contained no publicly available genomes, others with only a handful. The resulting genome collection is representative of samples around the world and across physiological conditions, contained many genomes from Candidate phyla radiation (CPR) which lack monoculture, and enabled discovery of new taxa such as a family within the Acholeplasmataceae order. New biomarkers were identified for rheumatoid arthritis or colorectal cancer, which would be more convenient than fecal samples. The large number of metagenomic samples also allowed assembly of many strains from important oral taxa such as Porphyromonas and Neisseria. Predicted functions enrich in drug metabolism and small molecule synthesis. Thus, these data lay down a genomic framework for future inquiries of the human oral microbiome.

microbiology↗

bioRxiv2019

A portable and cost-effective microfluidic system for massively parallel single-cell transcriptome profiling

Single-cell technologies are becoming increasingly widespread and have been revolutionizing our understanding of cell identity, state, diversity and function. However, current platforms can be slow to apply to large-scale studies and resource-limited clinical arenas due to a variety of reasons including cost, infrastructure, sample quality and requirements. Here we report DNBelab C4 (C4), a negative pressure orchestrated, portable and cost-effective device that enables high-throughput single-cell transcriptional profiling. C4 system can efficiently allow discrimination of species-specific cells at high resolution and dissect tissue heterogeneity in different organs, such as murine lung and cerebral cortex. Finally, we show that the C4 system is comparable to existing platforms but has huge benefits in cost and portability and, as such, it will be of great interest for the wider scientific community.

genomics↗

bioRxiv2019

A multi-tier species delimitation approach resolves conflicts in delineating the primitively segmented spider genus Heptathela endemic to Japanese islands

Determining species boundaries forms an important foundation for biological research. However, the results of molecular species delimitation can vary with the data sets and methods that are used. Here we use a two-step approach to delimit species in the genus Heptathela, a group of primitively segmented trapdoor spiders that are endemic to Japanese islands. Morphological evidence suggests the existence of 19 species in the genus. We tested this initial species hypothesis by using six molecular species-delimitation methods to analyse 180 mitochondrial COI sequences of Heptathela sampled from across the known range of the genus. We then conducted a set of more focused analyses by sampling additional genetic markers from the subset of taxa that were inconsistently delimited by the single-locus analyses of mitochondrial DNA. Multilocus species delimitation was performed using two Bayesian approaches based on the multispecies coalescent. Our approach identified 20 putative species among the 180 sampled individuals of Heptathela. We suggest that our two-step approach provides an efficient strategy for delimiting species while minimizing costs and computational time.

evolutionary biology↗