Biology subjects

Fang, Y.

Publications and source records attributed to Fang, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Cross regional coordination of neural activity in the human brain during autobiographical self-referential processing

For the human mind to operate, populations of neurons across remote regions of the brain need to coordinate their activity in the subsecond temporal scale. To date, our knowledge of such fast interactions involving cortical and subcortical structures in large brains, such as the human brain, remains limited. Here, we used stereo-electroencephalography (sEEG) recordings across four brain regions that are known, from decades of work, to be important for autobiographical memory processing. Our recordings involved 31 human participants implanted with intracranial electrodes in the hippocampus (HPC), posteromedial cortex (PMC), and ventromedial, as well as orbital subregions of the prefrontal cortex (OFC). In 14 subjects, we also recorded simultaneously in the anterior thalamus (ANT) across various experimental conditions and with direct electrical stimulations. Our observations provide new lines of correlative and causal evidence about the spatiotemporal profile of oscillatory coordination of cortical and subcortical activity during self-referential memory-based processing.

neuroscience↗

bioRxiv2023

AraENCODE: a comprehensive epigenomic database of Arabidopsis Thaliana

Arabidopsis (Arabidopsis thaliana) is a vital model organism in plant biology and genetics. In the last two decades, researchers have made significant progresses in characterizing the chromatin conformation and epigenomic information within the Arabidopsis genome. This information includes but is not limited to the higher structure of chromosomes, histone modification, DNA methylation, and chromatin accessibility. The results of these studies have provided an additional layer of information that complements the DNA sequence data. However, utilizing such knowledge poses a challenge for certain groups that lack bioinformatics analysts or adequate computing resources. A user-friendly and reproducible platform for accessing this information is urgently needed. In this study, we have developed a comprehensive epigenomic database for Arabidopsis (AraENCODE http://glab.hzau.edu.cn/AraENCODE), which comprises a total of 4,511 data libraries, including published chromatin conformation capture datasets (Hi-C, HiChIP), epigenomic datasets (ChIP-Seq, ATAC-Seq, FAIRE-Seq, MNase-Seq, DNase-Seq, BS-seq), and transcriptome data (RNA-Seq, miRNA-Seq). Furthermore, we have incorporated various existing resources, such as single nucleotide polymorphisms (SNPs), cis-regulatory modules, and multi-omics associations. We aim to provide a novel platform for investigating the regulation of epigenetic and chromatin interactions in Arabidopsis in relation to biological processes.

bioinformatics↗

bioRxiv2023

Comparative analysis of drug-like EP300/CREBBP acetyltransferase inhibitors

The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.

biochemistry↗

bioRxiv2023

The trade-off between individual metabolic specialization and versatility determines the metabolic efficiency of microbial communities

In microbial systems, a metabolic pathway can be either completed by one autonomous population or alternatively be distributed among a consortium performing metabolic division of labor (MDOL), where several specialized populations cooperate to complete the pathway. MDOL facilitates the systems function by reducing the metabolic burden; however, it may also hinder the function by reducing the exchange efficiency of metabolic intermediates among individuals. As a result, the metabolic efficiency of a community is influenced by trade-offs between the metabolic specialization and versatility of individuals, with the latter potentially introducing metabolic redundancy into the community. However, it remains unclear how metabolic specialization and versatility of the individuals involved can be controlled in order to optimize the function of the community. In this study, we deconstructed the metabolic pathway of naphthalene degradation into four specialized steps and introduced them individually or combinatorically into different strains, with varying levels of metabolic specialization. Using these strains, we engineered 1,456 synthetic consortia with varying levels of metabolic redundancy and tested their naphthalene degradation efficiency. We found that 74 consortia possessing metabolic redundancy exhibited higher degradation efficiency than both the autonomous population and the rigorous MDOL community. Quantitative modeling derived from our experiments provides general strategies for identifying the most effective MDOL consortium with functional redundancy (MCFR) from a range of possible MCFRs. Our large-scale genomic analysis suggests that natural communities for hydrocarbon degradation are mostly functionally redundant. In summary, our study provides critical insights into the engineering of high-performance microbial systems and explains why functional redundancy is prevalent in natural microbial communities.

microbiology↗

bioRxiv2023

Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice

Interspecies organogenesis via blastocyst complementation provides a unique platform to study development in an evolutionarily context and holds potential to overcome world-wide organ shortages1. By using this technique, rat pancreas, thymus, heart, and eye tissues have been generated in mice2-4. To date, however, xeno-generation of brain tissues has not been achieved through blastocyst complementation. Here, we developed an optimized one-step blastocyst complementation strategy based on C-CRISPR5, which facilitated rapid screening of candidate genes to support blastocyst complementation. Among the seven WNT pathway-related genes selected for targeting, only Dkk1 or Hesx1 deficiency supported forebrain complementation by blastocyst injection of mouse embryonic stem cells (mESCs). Further, injecting rat ESCs (rESCs) into mouse blastocysts deficient for Hesx1 but not Dkk1 supported the development of adult chimeric forebrains comprised a large proportion of rat cells that were structurally and functionally similar to the mouse forebrains. Our analysis revealed that the rESC-derived forebrains developed along the spatial-temporal trajectory with the mouse forebrains rather than rat forebrains, but gene expression profiles of rESC-derived nerve cells surprisingly maintained the characteristics of the rat cells. We noted that the chimeric rate gradually decreased as development progressed, suggesting xenogeneic barriers during mid-to-late prenatal development. Interspecies forebrain complementation opens the door for studying evolutionarily conserved and divergent mechanisms underlying brain development and cognitive function. The C-CRIPSR based IBC strategy developed here holds great potential to broaden the study and application of interspecies organogenesis.

developmental biology↗

bioRxiv2023

Duckweed evolution: from land back to water

Terrestialization is supposedly an important evolutionary process plant experience. However, directions of land back to water acquired little attention. Here we integrate multiproxy evidence to elucidate the evolution of duckweed. Three genera of duckweed show chronologically gradient degeneration in roots structure and stomatal function and decrease in lignocellulose content, accompanied by gradual contraction in relevant gene numbers and/or decline in transcription. The gene numbers in the main phytohormonal pathway are also gradually decreased. The co-action of auxin and rhizoid development gene causes a gradual decrease in adventitious roots. The significant expansion of the flavonoid pathway is also related to the adaptation of duckweed to floating growth. This study reconstructs the evolution history of duckweeds from land back to water, reverse to that of early land plants. SummaryWith terrestrialization being the popularly acknowledged plant evolutionary process, little is known about the evolution of higher plant from land back to water. Here we integrate multiproxy evidence to elucidate the gradual reverse evolution of duckweed. Three genera of duckweed show chronologically gradient degeneration in the structure of roots, the function of stomata, and decrease in lignocellulose content, accompanied by gradient contraction in relevant gene numbers and/or decline in transcript expression. The gene numbers in the main phytohormonal pathway are also gradually decreased. The co-action of auxin and rhizoid development gene causes a gradual decrease in adventitious roots. The significant expansion of the flavonoid pathway is also highly related to the adaptation of duckweed to floating growth. Our study combined with the fossil evidence reconstruct the evolution history of duckweeds from land back to water, reverse to that of early land plants. This study reconstructed the process of how a land plant returns to water, a reverse evolutionary approach which is different from what we studied in textbook about plant terrestrialization. This finding could be helpful for us to deeply and widely understand the adaptation of plant to the environment, and to expand and deepen the knowledge of evolution theory.

evolutionary biology↗

bioRxiv2023

Artificially cultivated duckweed: a high-efficiency starch producer

The increasing demand for starch has been a social struggle. We report a new technology that efficiently produces starch from duckweed. Although Landoltia punctata has a dramatic contraction of gene families, its starch content and productivity reached 72.2% (dry basis) and 10.4 g m-2 d-1 in 10 days, equivalent to a yield of 38.0 t ha-1 y-1 under nutrient limitation and CO2 elevation treatment. Meanwhile, we also examined the mechanism of duckweeds high starch accumulation. This is exhibited in the regulation of DNA methylation and transcription factors as well as the significantly up-regulated transcription levels and the increased enzyme activities of key genes in starch biosynthesis. Meantime, while nitrogen redistribution was enhanced, sucrose biosynthesis and transportation, and lignocellulose biosynthesis were all reduced. These alterations led to a reduction in lignocellulose and protein content and ultimately an increase in an accumulation of starch in the chloroplast. This work demonstrates duckweeds potential of being a highly efficient starch producer.

plant biology↗

bioRxiv2023

A Novel HIF-2α/ARNT Signaling Pathway Protects Against Microvascular Dysfunction and heart failure After Myocardial Infarction

Myocardial infarction (MI) significantly compromises the integrity of the cardiac microvascular endothelial barrier, leading to enhanced leakage and inflammation that contribute to the progression of heart failure. While HIF2 is highly expressed in cardiac endothelial cells (ECs) under hypoxic conditions, its role in regulating microvascular endothelial barrier function during MI is not well understood. In this study, we utilized mice with a cardiac-specific deletion of HIF2, generated through an inducible Cre (Cdh5Cre-ERT2) recombinase system. These mice exhibited no apparent phenotype under normal conditions. However, following left anterior descending (LAD) artery ligation-induced MI, they showed increased mortality associated with enhanced cardiac vascular leakage, inflammation, worsened cardiac function, and exacerbated heart remodeling. These outcomes suggest a protective role for endothelial HIF2 in response to cardiac ischemia. Parallel investigations in human cardiac microvascular endothelial cells (CMVECs) revealed that loss of ecHif2 led to diminished endothelial barrier function, characterized by reduced tight-junction protein levels and increased cell death, along with elevated expression of IL6 and other inflammatory markers. These effects were substantially reversed by overexpressing ARNT, a critical dimerization partner for HIF2 during hypoxia. Additionally, ARNT deletion also led to increased CMVEC permeability. Interestingly, ARNT, rather than HIF2 itself, directly binds to the IL6 promoter to suppress IL6 expression. Our findings demonstrate the critical role of endothelial HIF2 in response to MI and identify the HIF2/ARNT axis as a transcriptional repressor, offering novel insights for developing therapeutic strategies against heart failure following MI.

cell biology↗

bioRxiv2023

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid (N) protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, a mAb-based bELISA was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition (PI) cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 6.95%, and 5.15%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with COVID-19-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCEAntibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

microbiology↗

bioRxiv2023

Cancer associated fibroblasts serve as an ovarian cancer stem cell niche through noncanonical Wnt5a signaling

Frequent relapse and chemoresistance cause poor outcome in ovarian cancer (OC) and cancer stem cells (CSCs) are important contributors. While most studies focus exclusively on CSCs, the role of the microenvironment in providing optimal conditions to maintain their tumor-initiating potential remains poorly understood. Cancer associated fibroblasts (CAFs) are a major constituent of the OC tumor microenvironment and we show that CAFs and CSCs are enriched following chemotherapy in patient tumors. CAFs significantly increased OC cell resistance to carboplatin. Using heterotypic CAF-OC cocultures and in vivo limiting dilution assay, we confirmed that the CAFs act by enriching the CSC population. CAFs were found to increase the symmetric division of CSCs as well as the dedifferentiation of bulk OC cells into CSCs. The effect of CAFs was limited to OC cells in their immediate neighborhood, which could be prevented by inhibiting Wnt. Analysis of single cell RNA-seq data from OC patients revealed that Wnt5a as the highest expressed Wnt in CAFs and that certain subpopulations of CAFs express higher levels of Wnt5a. We found that Wnt5a from CAFs activated a noncanonical Wnt signaling pathway involving the ROR2/PKC/CERB1 axis in the neighboring CSCs. While canonical Wnt signaling was predominant in interactions between cancer cells in patients, non-canonical Wnt pathway was activated by CAF-OC crosstalk. Treatment with a Wnt5a inhibitor sensitized tumors to carboplatin in vivo. Together, our findings demonstrate a novel mechanism of CSC maintenance by signals from the microenvironmental CAFs, which can be targeted to treat OC chemoresistance and relapse. Statement of significanceCAFs serve as CSC niche through a Wnt5a mediated noncanonical Wnt signaling. Disease relapse and development of chemoresistance is a major problem in OC, which can be potentially addressed by targeting Wnt5a.

cancer biology↗

bioRxiv2023

High immigration rates critical for establishing emigration-driven diversity in microbial communities

Unraveling the mechanisms governing the diversity of ecological communities is a central goal in ecology. While microbial dispersal (including the emigration and immigration process) constitutes an important ecological process, the coupling effects of dispersal and microbial competition in microbial diversity are poorly understood. Here, we investigated how microbial dispersal affects the diversity of microbial communities in the presence of inter-species competition, using a generalized Lotka-Volterra model in combination with experimental investigations. Our model shows that emigration reduces the diversity induced by immigration at low immigration rates. We surprisingly find that it increases the diversity of the community when the immigration rates cross a defined threshold, which we identified as Ineutral. We also found that at high immigration rates, emigration weakens the relative abundance of fast-growing species, and thus enhances the mass effect and increases the diversity. We experimentally confirmed this finding using cocultures of 20 bacterial strains isolated from the soil. Our model further showed that Ineutral exists over a wide range of species pool sizes, growth rates, and interspecies interactions, and decreases with the increasing of species pool size, growth rate, and interspecies interaction. Our work deepens the understanding of the effects of dispersal on diversity of natural communities.

ecology↗

bioRxiv2023

Coronary Artery Disease risk variant dampens the expression of CALCRL by reducing HSF binding to shear stress responsive enhancer in endothelial cells

Coronary artery disease (CAD) is one of the major causes of mortality worldwide. Recent genome-wide association studies have started to unravel the genetic architecture of the disease. Such efforts have identified Calcitonin receptor-like (CALCRL), an important mediator of the endothelial fluid shear stress response, associated with CAD risk variants. In this study we functionally characterized the non-coding regulatory elements carrying CAD risks SNPs and studied their role in the regulation of CALCRL expression in endothelial cells. We demonstrate that rs880890-harboring regulatory element exhibits high enhancer activity and significant allelic bias with A allele showing 40% more activity than G allele. We also observed that the A allele of rs880890 is favored over the G allele under shear stress. CRISPR deletion of rs880890-enhancer resulted in downregulation of CALCRL expression. EMSA further showed that heat shock factors are binding to the enhancer with a preference for A allele over the G allele. In line with this, HSF1 knockdown resulted in a significant decrease in CALCRL expression. CALCRL knockdown as well as variant perturbation experiments confirmed the role of CALCRL in the regulation of eNOS, apelin, angiopoietin, prostaglandins and endothelin-1 signaling pathways while demonstrating a significant decrease in cell proliferation and tube formation. Overall, our results demonstrate the existence of an endothelial-specific heat shock factor regulated transcriptional enhancer carrying a CAD risk SNP rs880890 that regulates CALCRL expression. Better understanding of CALCRL gene regulation and the role of SNPs in modulation of CALCRL expression could provide important steps towards understanding genetic regulation of shear stress signaling responses.

genetics↗

bioRxiv2023

MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), one of the leading hereditary kidney diseases, and Alzheimers disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide. For the first time, in our newly generated mouse model recapitulating human ADTKD-UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

cell biology↗

bioRxiv2022

DNA methylation entropy is associated with DNA sequence featuresand developmental epigenetic divergence

Epigenetic information defines tissue identity and is largely inherited in development through DNA methylation. While studied mostly for mean differences, methylation also encodes stochastic change, defined as entropy in information theory. Analyzing allelespecific methylation in 48 human tissue sample datasets, we find that methylation entropy is associated with specific DNA binding motifs, regulatory DNA, and CpG density. Then applying information theory to 42 mouse embryo methylation datasets, we find that time- and tissue-specific patterns of development are more strongly correlated with methylation entropy than with mean, and methylation entropy is associated with sequence and chromatin features conserved with human. Moreover, methylation entropy is directly related to gene expression variability in development, suggesting a role for epigenetic entropy in developmental plasticity.

genomics↗

bioRxiv2022

Decoding protein binding landscape on circular RNAs with base-resolution Transformer models

Circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) to modulate gene expression. To date, most computational methods for predicting RBP binding sites on circRNAs focus on circRNA fragments instead of circRNAs. These methods detect whether a circRNA fragment contains binding sites, but cannot determine where are the binding sites and how many binding sites are on the circRNA transcript. We report a hybrid deep learning-based tool, CircSite, to predict RBP binding sites at single-nucleotide resolution and detect key contributed nucleotides on circRNA transcripts. CircSite takes advantage of convolutional neural networks (CNNs) and Transformer for learning local and global representations of circRNAs binding to RBPs, respectively. We construct 37 datasets of RBP-binding circRNAs for benchmarking and the experimental results show that CircSite offers accurate predictions of RBP binding nucleotides and detects key subsequences aligning well with known binding motifs.

bioinformatics↗

bioRxiv2022

In silico network-based screening reveals candidates for endothelial dysfunction therapy

Endothelial dysfunction (ED) is a hallmark of cardiovascular (CV) disorders and influences their progression; however, there are currently no direct therapeutic targets, primarily due to the lack of knowledge regarding EDs molecular basis. We used a computational approach to identify candidate targets for ED treatment. We constructed an ED disease gene network by combining the integration of epigenomics (ATAC-seq and ChIP-seq-H3K27ac) and transcriptomics data (RNA-seq) from human aorta endothelial cells (HAEC) exposed to surrogates of primary CV risk factors using network propagation. We then used in silico perturbation to prioritise genes that could influence the ED network most when removed. This process resulted in identifying 17 key candidates for which chemical inhibitors are available. These are genes associated with ED and atherosclerosis, and drugs that target those genes have not yet been tested for the treatment of CV disorders. The EGLN3 target and its inhibitor displayed significant anti-inflammatory and antioxidant properties in ECs assessed using a high-content screening platform. These findings illustrate the potential of in silico knockouts to discover disease-specific candidate targets for drug development or repositioning.

systems biology↗

bioRxiv2022

Foxa2 lineage+ mesendoderm forms a competitive pulmonary mesenchyme niche crucial for generating the entire lungs

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Identifying the crucial lineage and the program for lung organogenesis could facilitate designing whole-lung bioengineering. Using lineage-tracing mice and human iPSC-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung agenesis phenotype in mice. Importantly, wild-type donor mouse iPSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during early chimeric lung development, resulting in efficient complementation of the nearly entire lung niche at the late stage of lung development. These results suggest that lung complementation based on the Foxa2 lineage is a unique model for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and provides crucial insights for designing new bioengineering strategies to generate whole lungs.

developmental biology↗

bioRxiv2022

Chromosome territory reorganization through artificial chromosome fusion is negligible to cell fate determination and mouse development.

SummaryChromosomes occupy discrete spaces in the interphase cell nucleus, called chromosome territory. The structural and functional relevance of chromosome territory remains elusive. We fused chromosome 15 and 17 in mouse haploid embryonic stem cells (haESCs), resulting in distinct changes of territories in the corresponding native chromosomes, but affecting little on gene expression, pluripotency and gamete functions of haESCs. The karyotype engineered haESCs were successfully implemented in cultivating heterozygous (2n=39) and homozygous (2n=38) mouse models. The mice containing fusion-chromosome are fertile, and their representative tissues and organs display no phenotypic abnormalities, suggesting mouse development unscathed. These results indicate that the mammalian chromosome architectures are highly resilient, and reorganization of chromosome territories can be readily tolerated during cell differentiation and mouse development.

molecular biology↗