Biology subjects

Johnston, J.

Publications and source records attributed to Johnston, J..

30 records · Page 2Linked to original sources

bioRxiv2023

Differential substrate affinity and catabolite repression enable preferential use of urea by ammonia-oxidizing bacteria

Four distinct lineages of ammonia-oxidizing microorganisms (AOM) collectively contribute to one of the largest nitrogen fluxes in the global nitrogen budget. AOM possess widely different specific affinities for ammonia, thought to determine their niche differentiation. Nevertheless, ammonia-oxidizing archaea and bacteria (AOA, AOB), and complete ammonia oxidizers (comammox) co-occur in soils, freshwater sediments, and aquifers, suggesting that other factors must drive their coexistence. Here, we show that representatives of four AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct competition for either substrate. The tested AOA and comammox species preferentially used ammonia over urea, while beta-proteobacterial AOB favored urea utilization, repressed ammonia transport in the presence of urea, and showed higher affinity for urea than ammonia, whereas gamma-proteobacterial AOB co-utilized both substrates. Stable isotope tracing, kinetics, and transcriptomics experiments revealed that both assimilation and oxidation of ammonia are transport-dependent. These results reveal novel mechanisms of nitrogen metabolism regulation and transporter-based affinity underlying the contrasting niche adaptation and coexistence patterns among the major AOM lineages.

microbiology↗

bioRxiv2023

Comammox bacterial preference for urea influences its interactions with aerobic nitrifiers.

While the co-existence of comammox bacteria with canonical nitrifiers is well documented in diverse ecosystems, there is still a dearth of knowledge about the mechanisms underpinning their interactions. Understanding these interaction mechanisms is important as they may play a critical role in governing nitrogen biotransformation in natural and engineered ecosystems. In this study, we tested the ability of two environmentally relevant factors (nitrogen source and availability) to shape interactions between strict ammonia and nitrite-oxidizing bacteria and comammox bacteria in continuous flow column reactors. The composition of inorganic nitrogen species in reactors fed either ammonia or urea was similar during the lowest nitrogen loading condition (1 mg-N/L), but higher loadings (2 and 4 mg-N/L) promoted significant differences in nitrogen species composition and nitrifier abundances. The abundance and diversity of comammox bacteria were dependent on both nitrogen source and loading conditions as multiple comammox bacterial populations were preferentially enriched in the urea-fed system. In contrast, their abundance was reduced in response to higher nitrogen loadings in the ammonia-fed system likely due to ammonia-based inhibition. The preferential enrichment of comammox bacteria in the urea-fed system could be associated with their ureolytic activity calibrated to their ammonia oxidation rates thus minimizing ammonia accumulation to inhibitory levels. However, an increased abundance of comammox bacteria was not associated with a reduced abundance of nitrite oxidizers in the urea-fed system while a negative correlation was found between them in the ammonia-fed system; the latter dynamic likely emerging from reduced availability of nitrite to strict nitrite oxidizers at low ammonia loading conditions. ImportanceNitrification is an essential biological process in drinking water and wastewater treatment systems for managing nitrogen and protecting downstream water quality. The discovery of comammox bacteria and their detection alongside canonical nitrifiers in these engineered ecosystems has made it necessary to understand the environmental conditions that regulate their abundance and activity relative to other better-studied nitrifiers. This study aimed to evaluate two important factors that could potentially influence the behavior of nitrifying bacteria, and therefore impact nitrification processes. Colum reactors fed with either ammonia or urea were systematically monitored to capture changes in nitrogen biotransformation and the nitrifying community as a function of influent nitrogen concentration, nitrogen source, and reactor depth. Our findings show that comammox bacterial abundance decreased and that of nitrite oxidizers increased with increased ammonia availability, while their abundance and diversity increased with increasing urea availability without driving a reduction in the abundance of canonical nitrifiers.

microbiology↗

bioRxiv2022

Systematic multi-trait AAV capsid engineering for efficient gene delivery

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that evenly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 89% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited high production yields, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment, relative to AAV9, in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

synthetic biology↗

bioRxiv2022

Comparing transmission reconstruction models with Mycobacterium tuberculosis whole genome sequence data

Pathogen genomic epidemiology is now routinely used worldwide to interrogate infectious disease dynamics. Multiple computational tools that reconstruct transmission networks by coupling genomic data with epidemiological modelling have been developed. The resulting inferences are often used to inform outbreak investigations, yet to date, the performance of these transmission reconstruction tools has not been compared specifically for tuberculosis, a disease process with complex epidemiology that includes variable latency periods and within-host heterogeneity. Here, we carried out a systematic comparison of seven publicly available transmission reconstruction tools, evaluating their accuracy in predicting transmission events in both simulated and real-world Mycobacterium tuberculosis outbreaks. No tool was able to fully resolve transmission networks, though both the single-tree and multi-tree input implementations of TransPhylo identified the most epidemiologically supported transmission events and the fewest false positive links. We observed a high degree of variability in the transmission networks inferred by each approach. Our findings may inform an end-users choice of tools in future tuberculosis transmission analyses and underscore the need for caution when interpreting transmission networks produced using probabilistic approaches.

bioinformatics↗

bioRxiv2021

Diurnal modulation of multivesicular release controls the efficiency of information transmission at a sensory synapse

Neuromodulators adapt sensory circuits to changes in the external world or the animals internal state and synapses are key control sites for such plasticity. Less clear is how neuromodulation alters the amount of information transmitted through the circuit. We investigated this question in the context of the diurnal regulation of visual processing in the retina of zebrafish, focusing on ribbon synapses of bipolar cells. We demonstrate that contrast-sensitivity peaks in the afternoon accompanied by a four-fold increase in the average Shannon information transmitted from an active zone. This increase reflects higher synaptic gain, lower spontaneous "noise" and reduced variability of evoked responses. Simultaneously, an increase in the probability of multivesicular events with larger information content increases the efficiency of transmission (bits per vesicle) by factors of 1.5-2.7. This study demonstrates the multiplicity of mechanisms by which a neuromodulator can adjust the synaptic transfer of sensory information.

neuroscience↗

bioRxiv2021

TRIB1 regulates tumour growth via controlling tumour-associated macrophage phenotypes and is associated with breast cancer survival and treatment response

Molecular mechanisms that regulate tumour-associated macrophage (TAM) phenotype and function are incompletely understood. Here, we show that the pseudokinase TRIB1 is highly expressed by TAMs in breast cancer and that its expression correlates with response to chemotherapy and patient survival. We used immune-competent murine models of breast cancer to characterise the consequences of altered (reduced or elevated) myeloid Trib1 expression on tumour growth and composition of stromal immune cells. We found that both overexpression and knockout of myeloid Trib1 promote tumour growth, albeit through distinct molecular mechanisms. Myeloid Trib1 deficiency resulted in an early accelearation of tumour growth, paired with a selective reduction in perivascular macrophage numbers in vivo and enhanced oncogenic cytokine expression in vitro. In contrast, elevated levels of Trib1 in myeloid cells led to an increase in mammary tumour volume at late stages, together with a reduction of NOS2 expressing macrophages and an overall reduction of these cells in hypoxic tumour regions. In addition, we show that myeloid Trib1 is a previously unknown, negative regulator of the anti-tumour cytokine IL-15 and that increased expression of myeloid Trib1 leads to reduced IL-15 levels in mammary tumours, with a consequent reduction in the number of T-cells, that are key to anti-tumour immune responses. Together, these results define the different roles of TRIB1 in human breast cancer and provide a mechanistic understanding for the importance of myeloid TRIB1 expression levels in the development of this disease. SignificanceTRIB1 expression is strongly associated with response to chemotherapy in breast cancer patients with aggressive tumours. This protein is also highly expressed by tumour-associated macrophages. Thus, we used myeloid-specific alterations of Trib1 expression in mice (Trib1mKO and Trib1mTg), and characterised consequent changes in the growth rate and tumour microenvironment of mammary tumours. Both Trib1mKO and Trib1mTg enhanced tumour growth, but at different stages of tumour growth and via distinct mechanisms. Trib1mKO significantly increased the expression of oncogenic cytokines, such as IL6, IL10, CCL20, PD-L1, and VEGF. In contrast, Trib1mTg accelerated at the later stage of tumour growth via inhibition of hypoxic TAMs in the TME, as well as by reduced IL-15 expression thus leading to impaired naive and cytotoxic T cell infiltration. These data define TRIB1 as a potential novel marker of therapeutic responses in breast cancer, as well as a key mechanistic regulator of the anti-tumour cytokine, IL-15 in myeloid cells.

cancer biology↗

bioRxiv2020

Graded calcium spikes differentially signal neurotransmitter input in cerebrospinal fluid contacting neurons of mouse spinal cord

The action potential and its all-or-none nature is fundamental to neural communication. Canonically the action potential is initiated once voltage-gated Na+ channels are activated, and their rapid kinetics of activation and inactivation give rise to the action potentials all-or-none nature. Here we show that cerebrospinal fluid contacting neurons (CSFcNs) surrounding the central canal of the mouse spinal cord employ a different strategy. Rather than using voltage-gated Na+ channels to generate binary spikes, CSFcNs use two different types of voltage-gated Ca2+ channel, enabling spikes of different amplitude. T-type Ca2+ channels generate small amplitude spikes, whereas large amplitude spikes require high voltage-activated Cd2+ sensitive Ca2+ channels. We show that these different amplitude spikes signal input from different transmitter systems; purinergic inputs evoke smaller T-type dependent spikes while cholinergic inputs evoke large T-type independent spikes. Different synaptic inputs to CSFcNs can therefore be signalled by the spike amplitude.

neuroscience↗

bioRxiv2020

TATA and paused promoters active in differentiated tissues have distinct expression characteristics

Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this difference affects their tissue-specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active at all stages of development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late-stage Drosophila embryos to analyze the properties of promoter types. Using tissue-specific Pol II ChIP-seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. This difference between promoter types is associated with different chromatin accessibility in ATAC-seq data and different expression characteristics in single-cell RNA data. The results suggest that promoter types have optimized different promoter properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue-specific effector genes have evolved to use two different strategies for their differential expression across tissues.

genomics↗

bioRxiv2020

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (G)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for G biogenesis and membrane localization2, 3. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)4. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation5. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to G, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gi1 (isoform 1 of G family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of G, disrupting all three switch regions that convey G nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of G. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of G that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes G-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

biochemistry↗

bioRxiv2019

Myeloid Tribbles 1 induces early atherosclerosis via enhanced foam cell expansion

Macrophages drive atherosclerotic plaque progression and rupture, hence attenuating their atherosclerosis-inducing properties holds promise for reducing coronary heart disease (CHD). Recent studies in mouse models have demonstrated that Tribbles 1 (Trib1) regulates macrophage phenotype and shows that Trib1 deficiency increases plasma cholesterol and triglyceride levels, suggesting that reduced TRIB1 expression mediates the strong genetic association between the TRIB1 locus and increased CHD risk in man. However, we report here that myeloid-specific Trib1 (mTrib1) deficiency reduces early atheroma formation and that mTrib1 transgene expression increases atherogenesis. Mechanistically, mTrib1 increased macrophage lipid accumulation and the expression of a critical receptor (OLR1), promoting oxidized low density lipoprotein uptake and the formation of lipid-laden foam cells. As TRIB1 and OLR1 RNA levels were also strongly correlated in human macrophages, we suggest that a conserved, TRIB1-mediated mechanism drives foam cell formation in atherosclerotic plaque and that inhibiting mTRIB1 could be used therapeutically to reduce CHD.

immunology↗

bioRxiv2018

A retinal circuit generating a dynamic predictive code for orientated features

Sensory systems must reduce the transmission of redundant information to function efficiently. One strategy is to continuously adjust the sensitivity of neurons to suppress responses to common features of the input while enhancing responses to new ones. Here we image both the excitatory synaptic inputs and outputs of retinal ganglion cells to understand how such dynamic predictive coding is implemented in the analysis of spatial patterns. Synapses of bipolar cells become tuned to orientation through presynaptic inhibition generating lateral antagonism in the orientation domain. Individual ganglion cells receive excitatory synapses tuned to different orientations but feedforward inhibition generates a high-pass filter that only transmits the initial activation of these inputs, thereby removing redundancy. These results demonstrate how a dynamic predictive code can be implemented by circuit motifs common to many parts of the brain.

neuroscience↗

bioRxiv2018

Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-{kappa}B activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-{kappa}B at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

cell biology↗