Biology subjects

Wells, G.

Publications and source records attributed to Wells, G..

14 recordsLinked to original sources

bioRxiv2026

Dissecting KEAP1-NRF2 interactions: Structural and functional impact of cancer-associated KEAP1 mutants

Kelch-like ECH-associated protein 1 (KEAP1) is a cysteine-rich substrate adaptor for the CUL3-RBX1 E3 ubiquitin ligase complex, primarily regulating the transcription factor NRF2. By promoting NRF2 degradation under basal conditions, KEAP1 maintains redox homeostasis, while oxidative or electrophilic stress disrupts this regulation, enabling NRF2 activation of cytoprotective genes. Dysregulation of the KEAP1-NRF2 pathway, often through KEAP1 mutations, is implicated in cancer, where constitutive NRF2 activation promotes tumour growth, chemoresistance, and metabolic reprogramming. In this study, we investigated structural and functional consequences of three representative KEAP1 mutants R470C (superbinder), G480W (hypomorphic), and R554Q (null allele) using thermal shift assays (TSA), microscale thermophoresis (MST), isothermal calorimetry (ITC), and X-ray crystallography, complemented by AlphaFold3 modelling. TSA revealed that while inhibitor-induced thermal stability shifts followed similar pattern across mutants, baseline stability varied significantly, with R554Q showing the largest destabilisation. MST and ITC confirmed mutant-specific alterations in NRF2 ETGE peptide binding affinity, consistent with their known functional classifications. Structures of mutant-inhibitor and mutant-peptide complexes showed remarkable structural similarity to native KEAP1, with preserved inhibitor and peptide binding orientations despite residue substitutions. Spatial mapping of reported KEAP1 mutations revealed clustering of superbinder mutations near the IVR interface, while most loss-of-function variants localised within the KELCH domain. Our findings provide a detailed structural and biophysical characterization of clinically relevant KEAP1 mutations, offering mechanistic insights into their functional diversity. This work advances the understanding of KEAP1 mutation effects, with implications for therapeutic targeting of the KEAP1-NRF2 pathway in cancer.

cancer biology↗

bioRxiv2026

Glucose selectively drives a rapid oxidative burst and immunometabolic reprogramming in human neutrophils during Mycobacterium tuberculosis infection

Neutrophil functions have been linked to tuberculosis (TB)-associated tissue damage; however, the mechanisms driving immunopathology in the human TB lung remain poorly understood, due partly to the scarcity of human tissue for study. Here, we examine the metabolic and bioenergetic reprogramming of human neutrophils in response to Mycobacterium tuberculosis (Mtb) infection. In human necrotic TB granulomas, levels of NETosis-associated proteins are increased and co-localize with GLUT3, linking nutrient uptake to tissue damage. In vitro, Mtb elicits an immediate, contact-dependent oxidative burst in human neutrophils, and the magnitude of this response is carbon source-dependent. Glucose enables the most robust responses, indicating that glucose metabolism is a key driver of neutrophil-mediated inflammatory damage during TB. Mtb-induced responses are distinct from those induced by PMA, non-tuberculous mycobacteria, or other pathogenic intracellular bacteria, and are mediated through multiple neutrophil surface receptors. Notably, our data show that while the oxidative burst is carbon source-dependent, cytokine production is not. Further, Mtb infection reprograms neutrophil metabolism from glycolysis to the pentose phosphate pathway (PPP), generating NADPH required for the oxidative burst. Inhibiting G6PD, NADPH oxidase, or PAD4 significantly reduces this response, highlighting the PPP as a promising host target for mitigating TB immunopathology.

microbiology↗

bioRxiv2024

The KDM6 histone demethylase inhibitor GSK-J4 induces metal and stress responses in multiple myeloma cells

Thioneins are cysteine-rich apoproteins that regulate divalent metal homeostasis by virtue of their metal-chelation properties resulting in the ligand-bound metallothionein state. Previous studies show transient upregulation of the metallothionein (MT) gene cluster as part of a complex transcriptional response to a class of histone demethylase tool compounds targeting human Fe2+ dependent ketoglutarate oxygenases KDM6A (UTX) and KDM6B (JmjD3). The prototypic bioactive KDM6 inhibitor GSK-J4 induces apoptotic cell death in multiple myeloma cells and corresponding transcriptomic profiles are dominated by metal and metabolic stress response signatures, also observed in primary human myeloma cells. Here we investigate the hypothesis that metal-chelation by GSK-J4 provides the means for transport and intracellular release of Zn2+ leading to a metallothionein transcriptomic response signature. Live cell imaging of myeloma cells shows transient increases in intracellular free Zn2+ concentrations when exposed to GSK-J4, consistent with a model of inhibitor-mediated metal transport. Comparisons of GSK-J4 and ZnSO4 treatments in the presence or absence of metal chelators show that both treatment conditions induce different transcription factor repertoires with an overlapping MTF1 transcriptional regulation responsible for metallothionein and metal ion transport regulation. The data provide a possible explanation for the observed metal response upon GSK-J4 inhibition however the relationship with the pro-apoptotic mechanism in myeloma cells requires further investigation.

cell biology↗

bioRxiv2024

Basal activation of astrocytic Nrf2 in neuronal culture media: challenges and implications for neuron-astrocyte modelling

As a gatekeeper of anti-oxidant and anti-inflammatory cell protection, the transcription factor Nrf2 is a promising therapeutic target for several neurodegenerative diseases, leading to the development of Nrf2 activators targeting Keap1-dependent and independent regulatory mechanisms. Astrocytes play a crucial role in regulating neuronal physiology in health and disease, including Nrf2 neuroprotective responses. As neurons require specific conditions for their differentiation and maintenance, most 2D and 3D co-culture systems use medias that are compatible with neuronal differentiation and function, but also ensure astrocyte survival. Few studies, however, assess the molecular adaptations of astrocytes to changes from astrocyte maintenance medias alone, and their subsequent effects on neurons which may represent technical rather than physiological responses. Our findings show that while Nrf2 can be effectively activated by the Keap1-Nrf2 protein-protein interaction disruptor 18e, and classical Nrf2 activators DMF and CDDO-Me, in human primary cortical astrocyte monocultures, their efficacy is lost in LUHMES neuron-astrocyte co-cultures. Further investigation revealed that the Advanced DMEM/F12-based LUHMES differentiation media maximally induced basal Nrf2 activity in astrocytes alone, compared to astrocyte maintenance media, thus preventing pharmacological activation. Although Neurobasal slightly activated basal Nrf2, this was not significant suggesting that this media has less impact on astrocytic Nrf2 activity relative to Advanced DMEM/F12. As Nrf2 is a key regulator of oxidative damage and neuroinflammation, modelling these common features of neurodegenerative diseases may be confounded by environments that maximally activate basal Nrf2. Our findings thus suggest caution in media selection for neuron-astrocyte co-culture in disease modelling and therapeutic Nrf2 activator discovery.

neuroscience↗

bioRxiv2023

Detection of Mycobacterium tuberculosis in human tissue via RNA in situ hybridization

RationaleAccurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify Mycobacterium tuberculosis (Mtb) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of Mtb mRNA. ObjectivesTo employ RNAscope to determine the spatial distribution of Mtb mRNA within tuberculous human tissue, to appraise ZN-negative tissue from confirmed TB patients, and to provide proof-of-concept of RNAscope as a platform to inform TB diagnosis and Mtb biology. MethodsWe examined ante- and postmortem human TB tissue using RNAscope to detect Mtb mRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). Measurements and main resultsWe adapted RNAscope for Mtb and identified intact and disintegrated Mtb bacilli and intra- and extracellular Mtb mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. ConclusionsRNAscope has diagnostic potential and can guide therapeutic intervention as it detects Mtb mRNA and morphology in ZN-negative tissues from TB patients, and Mtb mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of Mtb bacilli exist in vivo.

microbiology↗

bioRxiv2023

Meta-omic profiling reveals ubiquity of genes encoding for the high value nitrogen rich biopolymer cyanophycin in activated sludge microbiomes

Recovering nitrogen (N) from municipal wastewater is a promising approach to prevent nutrient pollution, reduce energy use, and transition towards a circular N bioeconomy, but remains a technologically challenging endeavor. Existing N recovery techniques are optimized for high-strength, low-volume wastewater. Therefore, developing methods to concentrate dilute N from mainstream wastewater will bridge the gap between existing technologies and practical implementation. The N-rich biopolymer cyanophycin is a promising candidate for N bioconcentration and recovery due to its solubility characteristics and potential for high levels of accumulation in a limited number of bacterial isolates. However, the cyanophycin synthesis pathway is poorly explored in natural and engineered microbiomes. In this study, we analyzed over 3700 publicly available metagenome assembled genomes (MAGs) and found that the cyanophycin synthesis gene cphA was ubiquitous across common activated sludge bacteria. Surprisingly, we found that cphA was present in all analyzed genomes of the common phosphorus accumulating organisms (PAO) Ca. Accumulibacter and Tetrasphaera, suggesting potential for simultaneous N and P bioconcentration in the same organisms. Using metatranscriptomic data, we also confirmed the expression of cphA in lab-scale bioreactors enriched with PAO. Our findings suggest that cyanophycin synthesis is a ubiquitous metabolic pathway in activated sludge microbiomes and therefore may have potential for integration in existing biological nutrient removal and recovery processes. We anticipate this work to be a starting point for future evaluations of combined N and P bioaccumulation, with the ultimate goal of advancing widespread adoption of nutrient recovery from municipal wastewater.

ecology↗

bioRxiv2021

Intelligent Resolution: Integrating Cryo-EM with AI-driven Multi-resolution Simulations to Observe the SARS-CoV-2 Replication-Transcription Machinery in Action

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.

biophysics↗

bioRxiv2020

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design

Having claimed over 1 million lives worldwide to date, the ongoing COVID-19 pandemic has created one of the biggest challenges to develop an effective drug to treat infected patients. Among all the proteins expressed by the virus, RNA helicase is a fundamental protein for viral replication, and it is highly conserved among the coronaviridae family. To date, there is no high-resolution structure of helicase bound with ATP and RNA. We present here structural insights and molecular dynamics (MD) simulation results of the SARS-CoV-2 RNA helicase both in its apo form and in complex with its natural substrates. Our structural information of the catalytically competent helicase complex provides valuable insights for the mechanism and function of this enzyme at the atomic level, a key to develop specific inhibitors for this potential COVID-19 drug target.

biochemistry↗

bioRxiv2020

Integrated Omics Analyses Reveal Differential Gene Expression and Potential for Cooperation Between Denitrifying Polyphosphate and Glycogen Accumulating Organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N2O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The 6 transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA, and IC, and a competing glycogen accumulating organism (GAO) affiliated with Candidatus Competibacter. Strongly elevated expression of nitrite reductase compared to nitrous oxide reductase was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N2O accumulation. Surprisingly, while [~]90% of nitrite reductase gene transcripts mapped to the three co-occurring PAO populations, [~]93% of nitric oxide reductase gene transcripts were expressed by the GAO population. This suggests the potential for cooperation between GAOs and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide. Originality-Significance StatementPolyphosphate accumulating organisms (PAOs) affiliated with as-yet-uncultivated Ca. Accumulibacter phosphatis are increasingly employed in enhanced biological phosphorus removal (EBPR) processes, a common environmental biotechnology for removing phosphorus from wastewater and thereby preventing detrimental impacts of nutrient pollution. Under anoxic conditions, PAOs have been associated with unusually high emissions of the potent greenhouse gas and denitrification intermediate nitrous oxide. However, the underlying mechanisms and biological controls on incomplete denitrification by denitrifying Accumulibacter, their ecological interactions with understudied glycogen accumulating organisms (GAOs), and patterns of gene expression under anoxic conditions are all poorly understood. Here, we describe genomic features of a previously unrecognized clade of Accumulibacter that is putatively adapted to high rate P uptake under nitrite-driven denitrification and provide evidence that differential gene expression (namely elevated expression of nitrite reductase compared to nitrous oxide reductase) by Accumulibacter is a key control on nitrous oxide production. Moreover, we document genomic and transcriptional potential for cooperation and crossfeeding of the denitrification intermediate nitric oxide between GAOs and PAOs. This is surprising because GAOs are conventionally considered to be competitors to PAOs, and because nitric oxide is toxic to most microorganisms at low concentrations. Taken together, our work provides significant new understanding of metabolic and ecological interactions in EBPR processes that are critical to environmental protection; demonstrates the potential of previously unrecognized crossfeeding of the denitrification intermediate nitric oxide; and expands our understanding of genomic features and clade level diversity of Accumulibacter.

bioengineering↗

bioRxiv2019

The kinase inhibitor AT9283 selectively kills colorectal cancer cells with hyperactive NRF2

Aberrant hyperactivation of NRF2 is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis. The identification of ways to overcome the protection provided by NRF2 and selectively kill cancer cells addicted to NRF2 is a desirable goal. Exploiting the CRISPR/Cas9 technology, we generated colorectal cancer cell lines with hyperactive NRF2, and used them to perform a drug screen. We identified AT9283, an Aurora kinase inhibitor, for its selectivity towards killing cancer cells with hyperactive NRF2 as a consequence to either genetic or pharmacological activation. Our results show that hyperactivation of NRF2 presents a potential vulnerability that could be therapeutically exploited, and further suggest that AT9283, a drug that is currently in clinical trials, holds promise for the treatment of tumours with hyperactive NRF2.\n\nHighlightsO_LIWe present a new model for NRF2 hyperactivation in colorectal cancer cells.\nC_LIO_LIAT9283 selectively kills cancer cells with hyperactive NRF2\nC_LIO_LIBoth genetic and pharmacological activation of NRF2 sensitise cells to AT9283\nC_LI

cancer biology↗

bioRxiv2019

Integrated Low-Energy and Low Carbon Shortcut Nitrogen removal with Biological Phosphorus Removal for Sustainable Mainstream Wastewater Treatment

While enhanced biological phosphorus removal (EBPR) is widely utilized for phosphorus (P) removal from wastewater, understanding of efficient process alternatives that allow combined biological P removal and shortcut nitrogen (N) removal, such as nitritation-denitritation, is limited. Here, we demonstrate efficient and reliable combined total N, P, and chemical oxygen demand removal (70%, 83%, and 81%, respectively) in a sequencing batch reactor (SBR) treating real mainstream wastewater (primary effluent) at 20{degrees}C. Anaerobic - aerobic cycling (with intermittent oxic/anoxic periods during aeration) was used to achieve consistent removal rates, nitrite oxidizing organism (NOO) suppression, and high effluent quality. Importantly, high resolution process monitoring coupled to ex situ batch activity assays demonstrated that robust biological P removal was coupled to energy and carbon efficient nitritation-denitritation, not simultaneous nitrification-denitrification, for the last >400 days of 531 total days of operation. Nitrous oxide emissions of 2.2% relative to the influent TKN (or 5.2% relative to total inorganic nitrogen removal) were similar to those measured in other shortcut N bioprocesses. No exogenous chemicals were needed to achieve consistent process stability and high removal rates in the face of frequent wet weather flows and highly variable influent concentrations. Process modeling reproduced the performance observed in the SBR and confirmed that nitrite drawdown via denitritation contributed to suppression of NOO activity.

bioengineering↗

bioRxiv2018

Comammox Nitrospira are the dominant ammonia oxidizers in a mainstream low dissolved oxygen nitrification reactor

Recent findings show that a subset of bacteria affiliated with Nitrospira, a genus known for its importance in nitrite oxidation for biological nutrient removal applications, are capable of complete ammonia oxidation (comammox) to nitrate. Early reports suggested that they were absent or present in low abundance in most activated sludge processes, and thus likely functionally irrelevant. Here we show the accumulation of comammox Nitrospira in a nitrifying sequencing batch reactor operated at low dissolved oxygen (DO) concentrations. Actual mainstream wastewater was used as influent after primary settling and an upstream pre-treatment process for carbon and phosphorus removal. The ammonia removal rate was stable and exceeded that of the treatment plants parallel full-scale high DO nitrifying activated sludge reactor. 16S rRNA sequencing showed a steady accumulation of Nitrospira to 53% total abundance and a decline in conventional ammonia oxidizing bacteria to <1% total abundance over 400+ days of operation. After ruling out other known ammonia oxidizers, qPCR confirmed the accumulation of comammox Nitrospira beginning around day 200, to eventually comprise 94% of all detected amoA and 4% of total bacteria by day 407. Quantitative fluorescence in-situ hybridization confirmed the increasing trend and high relative abundance of Nitrospira. These results demonstrate that comammox can be metabolically relevant to nitrogen transformation in wastewater treatment, and can even dominate the ammonia oxidizing community. Our results suggest that comammox may be an important functional group in energy efficient nitrification systems designed to operate at low DO levels.

bioengineering↗

bioRxiv2018

Biomass segregation between biofilm and flocs improves the control of nitrite-oxidizing bacteria in mainstream partial nitritation and anammox processes

The control of nitrite-oxidizing bacteria (NOB) challenges the implementation of partial nitritation and anammox (PN/A) processes under mainstream conditions. The aim of the present study was to understand how operating conditions impact microbial competition and the control of NOB in hybrid PN/A systems, where biofilm and flocs coexist. A hybrid PN/A moving-bed biofilm reactor (MBBR; also referred to as integrated fixed film activated sludge or IFAS) was operated at 15 {degrees}C on aerobically pre-treated municipal wastewater (23 mgNH4-N{middle dot}L-1). Ammonium-oxidizing bacteria (AOB) and NOB were enriched primarily in the flocs, and anammox bacteria (AMX) in the biofilm. After decreasing the dissolved oxygen concentration (DO) from 1.2 to 0.17 mgO2{middle dot}L-1 - with all other operating conditions unchanged - washout of NOB from the flocs was observed. The activity of the minor NOB fraction remaining in the biofilm was suppressed at low DO. As a result, low effluent NO3- concentrations (0.5 mgN{middle dot}L-1) were consistently achieved at aerobic nitrogen removal rates (80 mgN{middle dot}L-1{middle dot}d-1) comparable to those of conventional treatment plants. A simple dynamic mathematical model, assuming perfect biomass segregation with AOB and NOB in the flocs and AMX in the biofilm, was able to qualitatively reproduce the selective washout of NOB from the flocs in response to the decrease in DO-setpoint. Similarly, numerical simulations indicated that flocs removal is an effective operational strategy to achieve the selective washout of NOB. The direct competition for NO2- between NOB and AMX - the latter retained in the biofilm and acting as a \"NO2-sink\" - was identified by the model as key mechanism leading to a difference in the actual growth rates of AOB and NOB (i.e., NOB < AOB in flocs) and allowing for the selective NOB washout. Experimental results and model predictions demonstrate the increased operational flexibility, in terms of variables that can be easily controlled by operators, offered by hybrid systems as compared to solely biofilm systems for the control of NOB in mainstream PN/A applications.\n\nHighlightsO_LIHybrid PN/A systems provide increased operational flexibility for NOB control\nC_LIO_LIAOB and NOB enrich primarily in the flocs, and AMX in the biofilm (\"NO2-sink\")\nC_LIO_LIAMX use NO2- allowing to differentiate AOB and NOB growth rates\nC_LIO_LIA decrease in DO or an increase in floc removal leads to selective NOB washout from flocs\nC_LIO_LIThe activity of the minor NOB fraction in the biofilm is suppressed at limiting DO\nC_LI\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC=\"FIGDIR/small/480780v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@cad5deorg.highwire.dtl.DTLVardef@9efef3org.highwire.dtl.DTLVardef@7409feorg.highwire.dtl.DTLVardef@3c1043_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2018

Epigenetic inactivation of oncogenic brachyury (TBXT) by H3K27 histone demethylase controls chordoma cell survival

The expression of the transcription factor brachyury (TBXT) is normally restricted to embryonic development and its silencing after mesoderm development is epigenetically regulated. In chordoma, a rare tumour of notochordal differentiation, TBXT acts as a putative oncogene, and we hypothesised that its expression could be controlled through epigenetic inhibition. Screening of five chordoma cell lines revealed that only inhibitors of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (Jmjd3) reduce TBXT expression and lead to cell death, findings validated in primary patient-derived culture systems. Pharmacological inhibition of KDM6 demethylases leads to genome-wide increases in repressive H3K27me3 marks, accompanied by significantly reduced TBXT expression, an effect that is phenocopied by the dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Transcriptional profiles in response to a novel KDM6A/B inhibitor, KDOBA67, revealed downregulation of critical genes and transcription factor networks for chordoma survival pathways, whereas upregulated pathways were dominated by stress, cell cycle and pro-apoptotic response pathways.\n\nThis study supports previous data showing that the function of TBXT is essential for maintaining notochord cell fate and function and provides further evidence that TBXT is an oncogenic driver in chordoma. Moreover, the data suggest that TBXT can potentially be targeted therapeutically by modulating epigenetic control mechanisms such as H3K27 demethylases.

cancer biology↗