Biology subjects

Gasser, R.

Publications and source records attributed to Gasser, R..

At least 19 recordsLinked to original sources

bioRxiv2025

New genomic resources for an invasive mite pest help guide the development of KASP markers for pesticide resistance screening

The redlegged earth mite, Halotydeus destructor, is an invasive pest of grain and pasture crops in Australia. Australian populations are increasingly developing resistance to broad-spectrum pesticides. Here, we assembled a chromosome-level genome for H. destructor, comprising nine chromosomes and totalling approximately 46 Mb. We also generated a genomic dataset from 190 populations collected across thousands of kilometres of its invasive Australian range and across four years (2018, 2021, 2022, and 2023), and used this dataset to develop KASP (Kompetitive allele-specific PCR) markers for identifying resistance mutations. We combined pooled whole-genome sequencing with resistance testing for organophosphate pesticides, chlorpyrifos and omethoate, and the pyrethroid pesticide bifenthrin. Target-site resistance to chlorpyrifos was strongly associated with the common ace G119S mutation. A second mutation, ace A201S, was also common, although its association with chlorpyrifos or omethoate resistance was less clear. Target-site resistance to bifenthrin was strongly associated with the common para L1024F mutation. We also detected several rare target-site mutations that likely contribute to resistance in specific populations, including F331Y in ace, and M918T, M918V, L925I, F1020S, and F1538I in para. Copy number variation in ace showed mixed associations with resistance, potentially reflecting the co-occurrence of variable gene copies and target-site mutations segregating within and among populations. Finally, we observed putative compositional and structural differences in genomic regions containing carboxylesterases with high ace-like homology, a group of genes that might be involved in organophosphate metabolism. This study advances the genomic and molecular resources available for H. destructor, and represents the most comprehensive investigation to date of resistance mechanisms and their spatiotemporal distribution in this invasive pest. Our data showed that pesticide resistance in H. destructor likely involves many genes. Nevertheless, target-site mutations are likely to remain key markers for rapid resistance diagnostics, for which our KASP markers will provide valuable tools.

genomics↗

bioRxiv2025

Comprehensive public health surveillance identifies novel human cryptosporidiosis subtypes and genetic diversity associated with swimming pool outbreaks in Australia, Victoria (2018-2024)

Cryptosporidium and Giardia are major causes of gastrointestinal illness globally. In Australia, cryptosporidiosis is a nationally notifiable disease, yet molecular characterisation of clinical cases is rarely performed, limiting the capacity to identify outbreaks, trace sources and assess zoonotic risk. During 2024 there was a 273% cases increase in Australia, the third country with the highest increase. We present the first comprehensive molecular investigation of human Cryptosporidium and Giardia infections in the state of Victoria, Australia. We analysed faecal samples collected between 2018 and 2024. Positive samples were subtyped and parasite load was estimated. Of the 2,330 samples tested, 225 were positive for Cryptosporidium and nine for Giardia. Seven Cryptosporidium species and 24 subtypes were identified, including multiple novel or regionally unique subtypes. C. hominis was the predominant species (85%), and three subtypes associated with 11 recreational water outbreaks in 2024. Based on spatiotemporal overlap and subtypes, 52 cases were inferred to represent undetected outbreak-associated infections. Several C. parvum subtypes reflected probable zoonotic transmission, two subtypes were associated with a childcare and camp outbreak. Six C. hominis subtypes and eight subtypes overall were reported for the first time in Australia. Globally novel subtypes of C. occultus, C. fayeri and C. meleagridis in human hosts is reported. The study reveals high diversity of Cryptosporidium subtypes in human infections in Victoria. This study demonstrates the potential for molecular surveillance to inform public health interventions when integrated with epidemiological data. These findings support One Health approaches to outbreak detection and source attribution. IMPORTANCECryptosporidium is a nationally notifiable pathogen in Australia, yet routine genotyping is not performed, limiting outbreak detection and source attribution. This study represents the first large-scale molecular surveillance of human Cryptosporidium and Giardia infections in southern Australia. By integrating species- and subtype-level typing with epidemiological metadata, we demonstrate the substantial diversity of infective lineages, including several novel or zoonotic genotypes. Our findings highlight the critical role of molecular tools in tracking transmission pathways, supporting outbreak investigation and informing public health responses. These data provide a foundation for incorporating routine genotyping into national surveillance strategies for parasitic enteropathogens. This study presents the first epidemiological study of the 2024 outbreaks in Australia. The global comparison of cases during this period also highlights potential large-scale disease dynamic of public health importance.

molecular biology↗

bioRxiv2025

KcatNet: advancing genome-wide enzyme turnover number prediction through structural enzymatic characterization

Enzyme turnover numbers (Kcat) are fundamental kinetic constants that quantify enzymatic efficiency. Systematic studies of Kcat are essential for characterizing the mechanisms underlying proteomic composition and cellular metabolism. However, experimental measurements of Kcat remain limited and prone to noise. To address this, we present KcatNet, a geometric deep learning model designed for high-throughput prediction of Kcat in metabolic enzymes across all organisms, leveraging paired enzyme sequence and substrate representations. KcatNet consistently outperforms existing predictors, particularly for enzymes with high catalytic efficiency, and demonstrates strong generalization to enzymes that are dissimilar to those in the training set. Furthermore, KcatNet uncovers structural mechanisms and interaction patterns within enzyme-substrate complexes, providing insights into architectural principles that are inaccessible with existing methods by harnessing the representational power of large-scale protein language models. We applied KcatNet to genome-scale Kcat prediction across diverse yeast species, improving proteome allocation predictions by integrating its outputs into metabolic models. Experimental validation further confirmed the models ability to identify enzyme mutants with enhanced activity. By bridging the gap between sequence, structure, and function, KcatNet establishes a robust foundation for advancing understanding of molecular-level mechanisms and accelerating enzyme engineering efforts.

bioinformatics↗

bioRxiv2024

SCREEN: a graph-based contrastive learning tool to infer catalytic residues and assess mutation tolerance in enzymes

The accurate identification of catalytic residues contributes to our understanding of enzyme functions in biological processes and pathways. The increasing number of protein sequences necessitates computational tools for the automated prediction of catalytic residues in enzymes. Here, we introduce SCREEN, a graph neural network for the high-throughput prediction of catalytic residues via the integration of enzyme functional and structural information. SCREEN constructs residue representations based on spatial arrangements and incorporates enzyme function priors into such representations through contrastive learning. We demonstrate that SCREEN (i) consistently outperforms currently-available predictors; (ii) provides accurate results when applied to inferred enzyme structures; and (iii) generalizes well to enzymes dissimilar from those in the training set. We also show that the putative catalytic residues predicted by SCREEN mimic key structural and biophysical characteristics of native catalytic residues. Moreover, using experimental data sets, we show that SCREENs predictions can be used to distinguish residues with a high mutation tolerance from those likely to cause functional loss when mutated, indicating that this tool might be used to infer disease-associated mutations.

bioinformatics↗

bioRxiv2024

scRCA: a Siamese network-based pipeline for the annotation of cell types using imperfect single-cell RNA-seq reference data

A critical step in the analysis of single-cell transcriptomic (scRNA-seq) data is the accurate identification and annotation of cell types. Such annotation is usually conducted by comparative analysis with known (reference) data sets - which assumes an accurate representation of cell types within the reference sample. However, this assumption is often incorrect, because factors, such as human errors in the laboratory or in silico, and methodological limitations, can ultimately lead to annotation errors in a reference dataset. As current pipelines for single-cell transcriptomic analysis do not adequately consider this challenge, there is a major demand for a computational pipeline that achieves high-quality cell type annotation using imperfect reference datasets that contain inherent errors (often referred to as "noise"). Here, we built a Siamese network-based pipeline, termed scRCA, that achieves an accurate annotation of cell types employing imperfect reference data. For researchers to decide whether to trust the scRCA annotations, an interpreter was developed to explore the factors on which the scRCA model makes its predictions. We also implemented 3 noise-robust losses-based cell type methods to improve the accuracy using imperfect dataset. Benchmarking experiments showed that scRCA outperforms the proposed noise-robust loss-based methods and methods commonly in use for cell type annotation using imperfect reference data. Importantly, we demonstrate that scRCA can overcome batch effects induced by distinctive single cell RNA-seq techniques. We anticipate that scRCA (https://github.com/LMC0705/scRCA) will serve as a practical tool for the annotation of cell types, employing a reference dataset-based approach.

bioinformatics↗

bioRxiv2023

Structure-function Analyses Reveal Key Molecular Determinants of HIV-1 CRF01_AE Resistance to the Entry Inhibitor Temsavir

The HIV-1 entry inhibitor temsavir prevents CD4 from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir-resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveal that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad-antiviral activity.

molecular biology↗

bioRxiv2022

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

microbiology↗

bioRxiv2022

Small CD4 mimetics sensitize HIV-1-infected macrophages to antibody-dependent cellular cytotoxicity

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

microbiology↗

bioRxiv2021

Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen

Spacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting did not increase magnitude of CD4+ T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.

immunology↗

bioRxiv2021

Detection of the HIV-1 accessory proteins Nef and Vpu by flow cytometry represents a new tool to study their functional interplay within a single infected CD4+ T cell

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4+ T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4+ T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4+ T cells. IMPORTANCEHIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate the lack of a functional Nef, which has implication for studies that use Nef-defective viruses to study ADCC responses.

microbiology↗

bioRxiv2021

An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

microbiology↗

bioRxiv2021

Across functional boundaries: making non-neutralizing antibodies to neutralize HIV-1 and mediate Fc-mediated effector killing of infected cells

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or Cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, closed Env trimer present on HIV-1 virions or expressed on HIV-1 infected cells. Antibodies targeting this region are therefore non-neutralizing and their potential as mediators of antibody depended cellular cytoxicity (ADCC) of HIV-1 infected cells diminished by a lack of available binding targets. Here we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or Cluster A specificity to the extracellular domain 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening closed Env trimers on HIV-1 particles/infected cells to expose the Cluster A region and activate ADCC and neutralization against these non-neutralizing targets. ImportanceHighly conserved epitopes within the co-receptor binding site (CoRBS) and constant region 1 and 2 (C1C2 or Cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4, therefore they are not accessible on virions and infected cells where the expression of CD4 is downregulated. Here we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or Cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1 infected cells through antibody depended cellular cytoxicity (ADCC) and inactivate HIV-1 virus through neutralization.

immunology↗

bioRxiv2021

Contribution of single mutations to selected SARS-CoV-2 emerging variants Spike antigenicity

Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37{degrees}C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.

microbiology↗

bioRxiv2021

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

microbiology↗

bioRxiv2021

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

microbiology↗

bioRxiv2021

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

immunology↗

bioRxiv2021

A single BNT162b2 mRNA dose elicits antibodies with Fc-mediated effector functions and boost pre-existing humoral and T cell responses

The standard dosing of the Pfizer/BioNTech BNT162b2 mRNA vaccine validated in clinical trials includes two doses administered three weeks apart. While the decision by some public health authorities to space the doses because of limiting supply has raised concerns about vaccine efficacy, data indicate that a single dose is up to 90% effective starting 14 days after its administration. We analyzed humoral and T cells responses three weeks after a single dose of this mRNA vaccine. Despite the proven efficacy of the vaccine at this time point, no neutralizing activity were elicited in SARS-CoV-2 naive individuals. However, we detected strong anti-receptor binding domain (RBD) and Spike antibodies with Fc-mediated effector functions and cellular responses dominated by the CD4+ T cell component. A single dose of this mRNA vaccine to individuals previously infected by SARS-CoV-2 boosted all humoral and T cell responses measured, with strong correlations between T helper and antibody immunity. Neutralizing responses were increased in both potency and breadth, with distinctive capacity to neutralize emerging variant strains. Our results highlight the importance of vaccinating uninfected and previously-infected individuals and shed new light into the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support to spacing the doses of two-vaccine regimens to vaccinate a larger pool of the population in the context of vaccine scarcity against SARS-CoV-2.

immunology↗

bioRxiv2021

Longitudinal analysis of humoral immunity against SARS-CoV-2 Spike in convalescent individuals up to 8 months post-symptom onset

Functional and lasting immune responses to the novel coronavirus (SARS-CoV-2) are currently under intense investigation as antibody titers in plasma have been shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we sought to determine the presence of SARS-CoV-2-specific memory B cells in COVID-19 convalescent patients. In this study, we report on the evolution of the overall humoral immune responses on 101 blood samples obtained from 32 COVID-19 convalescent patients between 16 and 233 days post-symptom onset. Our observations indicate that anti-Spike and anti-RBD IgM in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity in convalescent plasma declines rapidly compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which increase over time, and the number of IgG+ memory B cells which remain stable thereafter for up to 8 months after symptoms onset. With the recent approval of highly effective vaccines for COVID-19, data on the persistence of immune responses are of central importance. Even though overall circulating SARS-CoV-2 Spike-specific antibodies contract over time during convalescence, we demonstrate that RBD-specific B cells increase and persist up to 8 months post symptom onset. We also observe modest increases in RBD-specific IgG+ memory B cells and importantly, detectable IgG and sustained Fc-effector activity in plasma over the 8-month period. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for the prevention of secondary infections, vaccine efficacy and herd immunity against COVID-19.

microbiology↗