Biology subjects

Cao, L.

Publications and source records attributed to Cao, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Arp2/3-mediated bidirectional actin assembly by SPIN90 dimers in metazoans

Branched actin networks nucleated by the Arp2/3 complex play critical roles in various cellular processes, from cell migration to intracellular transport. However, when activated by WISH/DIP/SPIN90 family proteins, Arp2/3 nucleates linear actin filaments. Unexpectedly, we found that human SPIN90 is a dimer that can nucleate bidirectional actin filaments. To understand the basis for this, we determined a 3 [A] resolution structure of human SPIN90-Arp2/3 complex nucleating actin filaments. Our structure shows that SPIN90 dimerises via a 3-helix bundle and interacts with two Arp2/3 complexes. Each SPIN90 molecule binds both Arp2/3 complexes to promote their activation. Our analysis demonstrates that single filament nucleation by Arp2/3 is mechanistically more like branch formation than previously appreciated. The dimerisation domain in SPIN90 orthologues is conserved in metazoans, suggesting that this mode of bidirectional nucleation is a common strategy to generate anti-parallel actin filaments.

biophysics↗

bioRxiv2025

RegFormer: A Single-Cell Foundation Model Powered by Gene Regulatory Hierarchies

Single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of cellular diversity, but current computational models often fail to incorporate regulatory priors, handle data sparsity, or efficiently process long gene sequences. Here, we present RegFormer, a foundation model that integrates gene regulatory networks (GRNs) with Mamba-based state-space modeling, overcoming the scalability and context-length limitations of Transformer architectures. RegFormer encodes each gene through dual embeddings, a value embedding for quantitative expression and a token embedding for regulatory identity, organized within a GRN-guided gene order to capture both expression dynamics and hierarchical regulation. Pretrained on 26 million human single cells spanning 45 tissues and diverse biological contexts, RegFormer achieves superior scalability and biological fidelity. Across comprehensive benchmarks, it consistently outperforms state-of-the-art single-cell foundation models (scGPT, Geneformer, scFoundation, and scBERT), delivering higher clustering accuracy, improved batch integration, and more precise cell type annotation. RegFormer also reconstructs biologically coherent GRNs, accurately models transcriptional responses to genetic perturbations, and enhances drug response prediction across cancer cell lines. By combining regulatory priors with efficient long-sequence Mamba modeling, RegFormer establishes a biologically grounded and scalable framework for single-cell representation learning, enabling deeper mechanistic insight into gene regulation and cellular state transitions.

bioinformatics↗

bioRxiv2025

TRIM21-NUP98 interface accommodates structurally diverse molecular glue degraders

Molecular glue degraders enable targeted protein degradation by bridging interactions between target proteins and E3 ubiquitin ligases. Whereas some protein-protein interfaces exhibit the capacity to accommodate structurally diverse degraders, the extent of this adaptability across molecular glue targets remains unclear. We recently identified (S)-ACE-OH as a molecular glue degrader that recruits the E3 ubiquitin ligase TRIM21 to the nuclear pore complex by recognizing NUP98, thereby inducing the degradation of nuclear pore proteins. Here, we analyzed public compound toxicity data across a large collection of cell lines and identified two additional molecular glue degraders, PRLX 93936 and BMS-214662, that engage the TRIM21-NUP98 interface to induce the degradation of nucleopore proteins. Additionally, we confirmed that HGC652, another TRIM21-dependent molecular glue degrader, also binds at this interface. Together with our previously characterized degrader (S)-ACE-OH, these findings demonstrate that the TRIM21-NUP98 interface can accommodate structurally diverse molecular glue degraders, expanding the potential for its therapeutic exploitation.

biochemistry↗

bioRxiv2024

A Novel Bispecific Antibody CVL006 Superior to AK112 for Dual Targeting of PD-L1 and VEGF in Cancer Therapy

Both preclinical and clinical studies have shown that combining anti-VEGF/VEGFR drugs with immune checkpoint inhibitors (ICIs) significantly enhances anticancer efficacy. Currently, PDL-1/ VEGF bispecific antibodies demonstrate superior antitumor activity compared to monotherapy or even the combination of PD-L1 inhibitors with anti-VEGF antibodies. This enhanced efficacy results from the simultaneous blockade of the PD-1/PD-L1 pathway and the inhibition of VEGF-driven angiogenesis. In this study, we developed a novel bispecific antibody, CVL006, by fusing an anti-PDL1 VHH domain with a humanized IgG1 anti-VEGF monoclonal antibody while retaining antibody-dependent cellular cytotoxicity (ADCC) functionality. CVL006 demonstrated high affinity and specificity for both human PD-L1 and VEGF, effectively blocking both the VEGF/VEGFR signaling pathway and the PD-L1/PD-1 axis. This dual blockade not only suppressed VEGF-induced angiogenesis but also reactivated T cells, increasing the secretion of cytokines essential for immune response. In vivo studies further indicated that CVL006 achieved superior antitumor efficacy compared to the PD-L1 inhibitor atezolizumab in mouse models, with greater tumor growth inhibition and reduced angiogenesis. To compare with approved bispecific antibody PD-1/VEGF AK112 (ivonescimab), CVL006 showed superior antitumor efficacy in vivo. These findings underscore the therapeutic potential of CVL006, which integrates immune checkpoint inhibition with disruption of tumor vascularization, offering a robust and comprehensive anticancer strategy.

pharmacology and toxicology↗

bioRxiv2024

A signal of temporal integration in the human auditory brain: psychological insights, EEG evidence, and clinical application

Temporal integration, the process by which the auditory system combines sound information over a curtain period to form a coherent auditory object, is essential for coherent auditory perception, yet its neural mechanisms remain underexplored. We use a "transitional click train" paradigm, which concatenates two click trains with slightly differing inter-click intervals (ICIs), to investigate temporal integration in the human cortex. Using a 64-channel electroencephalogram (EEG), we recorded responses from 42 healthy participants exposed to regular and irregular transitional click trains and conducted change detection tasks. Regular transitional click trains elicited significant change responses in the human cortex, indicative of temporal integration, whereas irregular trains did not. These neural responses were modulated by ICI length, ICI contrast, and regularity. Behavioral data mirrored EEG findings, showing enhanced detection for regular conditions compared to irregular conditions and pure tones. Furthermore, variations in change responses were associated with decision-making processes. Temporal continuity was critical, as introducing gaps between click trains diminished both behavioral and neural responses. In clinical assessments, 22 coma patients exhibited diminished or absent change responses, effectively distinguishing them from healthy individuals. Our findings identify distinct neural markers of temporal integration and highlight the potential of transitional click trains for clinical diagnostics.

neuroscience↗

bioRxiv2024

BioLLM: A Standardized Framework for Integrating and Benchmarking Single-Cell Foundation Models

The application and evaluation of single cell foundational models (scFMs) present significant challenges stemming from the heterogeneity of architectural frameworks and coding standards. To address these issues, we introduce BioLLM, a framework facilitating the integration and application of scFMs in single-cell RNA sequencing data analysis. BioLLM provides a universal interface, bridging diverse scFMs into a seamless ecosystem. By mitigating architectural disparities and coding conventions, it empowers researchers with streamlined access to scFMs. With standardized APIs and comprehensive documentation, BioLLM streamlines model switching and comparative analyses, while incorporating best practices for consistent model evaluation. Our comprehensive evaluation of scFMs revealed distinct strengths and limitations, highlighting scGPTs robust performance across all tasks, both in zero-shot and fine-tuning scenarios. Geneformer and scFoundation also demonstrated strong capabilities in gene-level tasks, benefiting from effective pretraining strategies. In contrast, scBERT underperformed relative to other models, likely attributable to its considerably smaller parameter count and the limited size of the training dataset. Ultimately, BioLLM aims to empower the scientific community to leverage the full potential of foundational models, advancing our understanding of complex biological systems through enhanced single-cell analysis.

bioinformatics↗

bioRxiv2024

Identification of Covalent Cyclic Peptide Inhibitors Targeting Protein-Protein Interactions Using Phage Display

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 Spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.

microbiology↗

bioRxiv2024

Cysimiditides: RiPPs with a Zn-tetracysteine motif and aspartimidylation

Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for expression. Cysimiditides bind a Zn2+ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn2+ is required for methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn2+-tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn2+-tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally-confirmed RiPPs harboring aspartimides, and represent the first RiPP class that has an obligate metal ion. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/616296v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@2879corg.highwire.dtl.DTLVardef@152b63org.highwire.dtl.DTLVardef@1d3ad25org.highwire.dtl.DTLVardef@c8d63_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Microglia replacement effectively attenuates the disease progress of ALSP in the mouse model and human patient

Microglia play critical roles in the brain physiology and pathology. CSF1R is primarily expressed in microglia. The mono-allelic CSF1R mutation causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a lethal neurological disease and no rational cure in clinical trials. There are no animal models mimicking human ALSP. In this study, we first developed mouse models based on human ALSP hotspot mutations. We then utilized microglia replacement by bone marrow transplantation (Mr BMT) to replace the Csf1r-deficient microglia in ALSP mice by Csf1r-normal donor cells. With pathogenic gene correction, Mr BMT efficiently attenuated the pathologies. Previously, an ALSP patient received traditional bone marrow transplantation (tBMT) due to a misdiagnosis of metachromatic leukodystrophy. The disease progress was halted for 15 years with unknown reasons. We demonstrated that tBMT in ALSP is equivalent to or close to Mr BMT, achieving efficient microglia replacement and therefore attenuating the ALSP progress in the mouse model. Next, we applied tBMT to replace CSF1R-deficient microglia in human patients. Our clinical results show that after microglia replacement, the ALSP course is effectively halted. Together, microglia replacement corrects the pathogenic gene and thus halts the disease progress in the mouse model and human patients. This study strongly demonstrates clinical potentials of microglia replacement in neurological disease treatments.

neuroscience↗

bioRxiv2024

Cold seeps are hotspots of deep-sea nitrogen-loss driven by microorganisms across 21 phyla

Nitrogen bioavailability, governed by the balance of fixation and loss processes, is a key factor regulating oceanic productivity, ecosystem functions, and global biogeochemical cycles. The key nitrogen-loss organisms--denitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria--are not well understood in marine seafloor environments, especially in deep-sea cold seeps. In this study, we combined geochemical measurements, 15N stable isotope tracer analysis, metagenomics, metatranscriptomics, and three-dimensional protein structural simulations to investigate the diversity of denitrifying and anammox microbial communities and their biogeochemical roles in these habitats. Geochemical evidence from 301 sediment samples shows significantly higher nitrogen-loss rates in cold seeps compared to typical deep-sea sediments, with an estimated annual nitrogen loss of 6.16 Tg from seafloor surface sediments. Examination of a total of 147 million non-redundant genes reveals a high prevalence and active expression of nitrogen-loss genes, including nitrous-oxide reductase (NosZ; 6.88 genes per million or GPM on average), nitric oxide dismutase (Nod; 1.29 GPM), and hydrazine synthase (HzsA; 3.35 GPM) in surface sediments. Analysis of 3,164 metagenome-assembled genomes from this habitat has expanded the known diversity of nitrous-oxide reducers to six phyla and nitric oxide-dismutating organisms to one phylum and two new orders, while ten phyla host anammox bacteria going beyond Planctomycetota. These microbes show diverse structural adaptations and complex gene cluster arrangements that potentially enable survival in the harsh conditions of cold seeps. These findings suggest that cold seeps, despite their low temperatures, are significant, previously underestimated hotspots of nitrogen loss, potentially contribute substantially to the global nitrogen cycle.

microbiology↗

bioRxiv2024

Design of High Affinity Binders to Convex Protein Target Sites

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGF{beta}RII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGF{beta}RII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

biochemistry↗

bioRxiv2024

Profiling trace organic chemical biotransformation genes, enzymes and associated bacteria in microbial model communities

Microbial biotransformation of trace organic chemicals (TOrCs) is an essential process in wastewater treatment for eliminating environmental pollution. Understanding of TOrC biotransformation mechanisms, especially at their original concentrations, is important to optimize treatment performance, whereas our current knowledge is limited. Here we investigated the biotransformation of seven TOrCs by 24 model communities. The genome-centric analyses unraveled the biotransformation drivers concerning functional genes and enzymes and responsible bacteria. We obtained efficient model communities for complete removal on ibuprofen, caffeine and atenolol, and the transformation efficiencies for sulfamethoxazole, carbamazepine, trimethoprim and gabapentin were 0-45%. Biotransformation performance was not fully reflected by the presence of known biotransformation genes and enzymes. However, functional similar homologs to existing biotransformation genes and enzymes (e.g., long-chain-fatty-acid-CoA ligase encoded by fadD and fadD13 gene, acyl-CoA dehydrogenase encoded by fadE12 gene) could play critical roles in TOrC metabolism. Finally, we identified previously undescribed degrading strains, e.g., Rhodococcus qingshengii for caffeine, carbamazepine, sulfamethoxazole and ibuprofen biotransformation, and potential transformation enzymes, e.g., SDR family oxidoreductase targeting sulfamethoxazole and putative hypothetical proteins for caffeine, atenolol and gabapentin biotransformation.

microbiology↗

bioRxiv2024

PROTAC-mediated dual degradation of BCL-xL and BCL-2 is a highly effective therapeutic strategy in small-cell lung cancer

BCL-xL and BCL-2 are validated therapeutic targets in small-cell lung cancer (SCLC). Targeting these proteins with navitoclax (formerly ABT263, a dual BCL-xL/2 inhibitor) induces dose-limiting thrombocytopenia through on-target BCL-xL inhibition in platelets. Therefore, platelet toxicity poses a barrier in advancing the clinical translation of navitoclax. We have developed a strategy to selectively target BCL-xL in tumors, while sparing platelets, by utilizing proteolysis-targeting chimeras (PROTACs) that hijack the cellular ubiquitin proteasome system for target ubiquitination and subsequent degradation. In our previous study, the first-in-class BCL-xL PROTAC, called DT2216, was shown to have synergistic antitumor activities when combined with venetoclax (formerly ABT199, BCL-2-selective inhibitor) in a BCL-xL/2 co-dependent SCLC cell line, NCI-H146 (hereafter referred to as H146), in vitro and in a xenograft model. Guided by these findings, we evaluated our newly developed BCL-xL/2 dual degrader, called 753b, in three BCL-xL/2 co-dependent SCLC cell lines and the H146 xenograft models. 753b was found to degrade both BCL-xL and BCL-2 in these cell lines. Importantly, it was considerably more potent than DT2216, navitoclax, or DT2216+venetoclax to reduce the viability of BCL-xL/2 co-dependent SCLC cell lines in cell culture. In vivo, 5 mg/kg weekly dosing of 753b leads to significant tumor growth delay similar to the DT2216+venetoclax combination in H146 xenografts by degrading both BCL-xL and BCL-2. Additionally, 753b administration at 5 mg/kg every four days induced tumor regressions. 753b at this dosage was well tolerated in mice without induction of severe thrombocytopenia as seen with navitoclax nor induced significant changes in mouse body weights. These results suggest that the BCL-xL/2 dual degrader could be an effective and safe therapeutic for a subset of SCLC patients warranting clinical trials in future.

cancer biology↗

bioRxiv2024

Proteus: pioneering protein structure generation for enhanced designability and efficiency

Diffusion-based generative models have been successfully employed to create proteins with novel structures and functions. However, the construction of such models typically depends on large, pre-trained structure prediction networks, like RFdiffusion. In contrast, alternative models that are trained from scratch, such as FrameDiff, still fall short in performance. In this context, we introduce Proteus, an innovative deep diffusion network that incorporates graph-based triangle methods and a multi-track interaction network, eliminating the dependency on structure prediction pre-training with superior efficiency. We have validated our models performance on de novo protein backbone generation through comprehensive in silico evaluations and experimental characterizations, which demonstrate a remarkable success rate. These promising results underscore Proteuss ability to generate highly designable protein backbones efficiently. This capability, achieved without reliance on pre-training techniques, has the potential to significantly advance the field of protein design. Codes are available at https://github.com/Wangchentong/Proteus.

bioinformatics↗

bioRxiv2024

Increased regional P2X7R expression detected by GSK1482160 PET in a tauopathy mouse model

Neuroinflammation plays an important role in Alzheimers disease and primary tauopathies. The aim of the current study was to map [18F]GSK1482160 for imaging of purinergic P2X7R in Alzheimers disease and primary tauopathy mouse models. MicroPET was performed using [18F]GSK1482160 in widely used mouse models of Alzheimers disease (APP/PS1, 5xFAD and 3xTg), 4-repeat tauopathy (rTg4510) mice and age-matched wild-type mice. Increased uptake of [18F]GSK1482160 was observed in the cortex and basal forebrain of 7-month-old rTg4510 mice compared to age-matched wild-type mice and compared to 3-month-old rTg4510 mice. Nonparametric Spearmans rank analysis revealed a positive correlation between tau [18F]APN-1607 uptake and [18F]GSK1482160 in the hippocampus of rTg4510 mice. No significant differences in the uptake of [18F]GSK1482160 were observed between wild-type mice and APP/PS1 mice (5, 10 months), 5xFAD mice (3, 7 months) or 3xTg mice (10 months). Immunofluorescence staining further indicated the distribution of P2X7Rs in the brains of 7-month-old rTg4510 mice with accumulation of tau inclusion compared to wild-type mice. These findings provide in vivo imaging evidence for increased P2X7R in the brains of tauopathy model mice.

neuroscience↗

bioRxiv2024

WRKY1 confers resistance to powdery mildew by accelerating SAR and preventing over-immunity in apple.

Powdery mildew is one of the most serious diseases in apple production. SAR has a broad-spectrum immunity in plants against pathogen. Plants activate SAR against pathogen invasion and also prevent over-immunity. The relevant mechanism is still unknown in apple. In this study, we isolated and identified powdery mildew pathogen from the field and preserved them on the apple tissue culture seedlings. We performed DAP-seq of powdery mildew-inducible WRKY40. WRKY40 positively regulates NPR3like by directly binding to the W-box element of its promoter. NPR3like represses the expression of the PR1 gene in the presence of SA by competing with TGA2 for binding to NPR1. WRKY1 positively regulates WRKY40 by directly binding to the dual W-box element of its promoter, while WRKY1 positively regulates NPR3like by directly binding to the W-box element of its promoter. The expression trends of WRKY1, WRKY40, and NPR3like were basically the same as that of PR1 within 24 h after powdery mildew and SA treatments. Besides, WRKY1 increased SA content by positively regulating EPS1. After inoculation with powdery mildew, the up-regulation of PR1 in RNAi-silenced plants of WRKY1 was more slowly compared with the wild type, and the number of spores and mycelium increased significantly. In summary, we established a new model of NPR3like inhibition of NPR1 activity positively regulated by the WRKY1-WRKY40 module and found that the WRKY1-EPS1 module accelerated the up-regulation of PR1 by increasing the SA content. Finally, we elucidated WRKY1 confers resistance to powdery mildew by accelerating SAR and preventing over-immunity in apple.

molecular biology↗

bioRxiv2024

Breaking free from the clocks tyranny restores memory to brain damaged flies

The relationship between sleep and memory is an active topic of investigation. In this context, we demonstrate that enhancing sleep restores memory to flies with ablated Mushroom Bodies (MB), a key memory center; this is consistent across several memory assays. Mapping the underlying circuitry reveals circadian modulation of a subset of Dopaminergic neurons (DANs) that modulate aversive learning. Using imaging, we show that MB-ablation disrupts, and sleep restores the time of day these neurons are most responsive. Knocking down the receptor for the clock output signal, Pigment-dispersing factor (Pdfr), in this subset of DANs restores memory to MB-ablated flies. Crucially, MB-ablation does not result in memory impairments in the absence of a functioning clock. Our results reveal neuromodulations key role in cognitive restoration, where sleep aids memory in damaged brains, but a functioning clock unexpectedly hinders this process.

neuroscience↗

bioRxiv2024

Phylogenetically and structurally diverse reductive dehalogenases link biogeochemical cycles in deep-sea cold seeps

Reductive dehalogenation is crucial for halogen cycling and environmental remediation, yet its ecological role is not completely understood, especially in deep-sea environments. To address this gap, we investigated the diversity of reductive dehalogenases (RDases) and ecophysiology of organohalide reducers in deep-sea cold seeps, which are environments rich in halogenated compounds. Through genome- resolved metagenomic analysis of 165 global cold seep sediment samples, we identified four types of RDases, namely prototypical respiratory, transmembrane respiratory, and cytosolic RDases, and one novel clade. Cold seeps were found to harbor a higher abundance of these RDase genes compared to other marine sediments, establishing them as unique hotspots for microbial reductive dehalogenation activity. These RDases are encoded by a wide range of microorganisms across four archaeal and 36 bacterial phyla, significantly expanding the known diversity of organohalide reducers. Halogen geochemistry, laboratory incubations with various halogenated compounds, metatranscriptomic data, and metabolomic profiling confirmed the presence of organohalides at concentrations of up to 18 mg/g in these sediments and demonstrated their active microbial reduction. This process is tightly linked to other biogeochemical cycles, including carbon, hydrogen, nitrogen, sulfur, and trace elements. RDases from cold seeps have diverse N-terminal structures across different gene groups, and reductive dehalogenase genes in these environments are mostly functionally constrained and conserved. These findings collectively suggest that reductive dehalogenation is a central process in deep-sea environments, mediated by a diverse array of microbes and novel enzymes.

microbiology↗