Biology subjects

Chauhan, V.

Publications and source records attributed to Chauhan, V..

At least 19 recordsLinked to original sources

bioRxiv2026

A Systems Framework for Quantifying Programmability and Persistence Across Mammalian Cell Types

Cellular therapies, toxicity screening, and regenerative medicine depend on selecting mammalian cell types with optimal lifespan, persistence post-transplant, immunogenicity, and chemical resilience. This review synthesizes data from over 50 immune, parenchymal, stem, and emerging engineered cell populations--including gamma-delta T cells, iNKT cells, CAR-macrophages, and hypoimmune iPSC derivatives--drawing from in vivo lifespan studies (including 1{blacksquare}C birth-dating and deuterium labeling), engraftment dynamics, immune rejection risk, and stress sensitivity profiles. We introduce a Programmability & Persistence Score (PPS; 0-20) that integrates these features into a unified metric, complemented by Pareto frontier analysis to visualize multi-objective trade-offs. High-PPS cell types (e.g., HLA-matched HSCs, hypoimmune iPSCs, chondrocytes) are suited for long-term regenerative applications, while low-PPS sentinels (e.g., neutrophils, enterocytes) serve acute assays. We discuss mathematical extensions including multi-criteria decision analysis, fuzzy membership functions, and Bayesian frameworks that address limitations of linear additive scoring. Together, these integrated profiles support cell selection for gene editing, organ-on-chip systems, in vivo cell programming, and immunotherapy, bridging cell biology with translational engineering.

systems biology↗

bioRxiv2026

Catalytic Efficiency of Conserved Metabolic Enzymes Across Evolutionary Complexity

Metabolic enzymes are highly conserved across life, yet their catalytic efficiencies vary among organisms of differing complexity. We compiled turnover numbers (kcat) and catalytic efficiencies (kcat/Km) for key enzymes in glycolysis, the TCA cycle, and oxidative phosphorylation from bacteria to mammals, drawing on BRENDA, BioNumbers, and primary literature. Rather than a universal trend, we find enzyme-specific and pathway-dependent patterns: TCA cycle dehydrogenases (citrate synthase, malate dehydrogenase, isocitrate dehydrogenase) consistently show 3-4x higher kcat in E. coli than in mammals, whereas glycolytic kinases and transferases show the opposite pattern--human RBC pyruvate kinase ([~]1,375 s-1) is 2.8x faster than E. coli ([~]497 s-1), and yeast GAPDH ([~]1,000 s-1) is 10x faster than bacterial. OXPHOS complexes (ATP synthase, cytochrome c oxidase) show remarkably conserved rates across kingdoms. We interpret these patterns through the lens of metabolic demand, regulatory complexity, and the "rapid bursts and slow declines" framework of enzyme evolution, arguing that context-specific selection pressures--not organismal complexity per se--determine enzyme kinetic tuning.

biochemistry↗

bioRxiv2026

Reprogramming BCMA-Targeted CAR-T Cells through γ-Secretase Modulation Blocks Antigen Shedding and Extends CAR-T Longevity

B-cell maturation antigen (BCMA) shedding by {gamma}-secretase generates soluble BCMA (sBCMA), which diminishes membrane antigen density, and limits the durability of BCMA-directed immunotherapies in multiple myeloma (MM). Here, using AI-driven diffusion modeling, we identify peptide inhibitors that selectively block {gamma}-secretase-mediated BCMA cleavage, stabilizing membrane-bound BCMA (mBCMA) without compromising cellular viability. The lead peptide, P5, suppresses sBCMA and restores mBCMA in vitro and in xenograft models. To create sustained, cell-intrinsic inhibition, we engineered Shedding Deterrent Locked-in (SHEDLOCK) CAR-T cells that locally secrete {gamma}-secretase-modulatory peptides. SHEDLOCK-1 CAR-T cells secreting P5 exhibits enhanced cytotoxicity and persistence relative to conventional CAR-T cells. Building on this, SHEDLOCK-2 CAR-T cells were generated, which secrete a modified, naturally derived peptide (nxP) that directly engages the {gamma}-secretase catalytic site, concurrently preventing BCMA shedding, and surprisingly enhancing CAR-T cell longevity by preserving telomere integrity and metabolic fitness. In MM xenograft and patient-derived models, SHEDLOCK-2 CAR-T cells demonstrate durable antitumor activity with a favorable safety profile. Together, these findings establish SHEDLOCK as a next-generation CAR-T platform and provide a strong preclinical foundation for Phase I clinical evaluation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/700575v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@3cd38aorg.highwire.dtl.DTLVardef@164167borg.highwire.dtl.DTLVardef@1ffed21org.highwire.dtl.DTLVardef@1294d06_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Thermal shifts can override the effects of Transcription-targeting Antibiotics

Bacteria traverse diverse stresses but rarely experience them in isolation. Thermal fluctuations and antibiotic exposure often coincide. Yet, how cells transduce these concurrent cues into gene regulatory programs remains unresolved. Here, we show that in Escherichia coli, cold and heat shocks exert dominance over antibiotic stresses across multiple levels of bacterial physiology. First, combined exposure to antibiotics and thermal shifts produced transcriptomes that consistently converged to temperature-defined transcriptomic states. This dominance emerges through the effects of thermal shifts on metabolism, nucleoid organization, engagement of antibiotic targets with DNA, over-expression of global transcription regulators, drug-target conformational plasticity, and regulatory induction of efflux pathways during combined exposure. Finally, cross-species simulations and phylogenetic analysis revealed that thermal disruption of antibiotic efficiency is conserved across evolutionary distant bacterial species. Together, these results identify temperature as a critical determinant of antibiotic efficacy.

microbiology↗

bioRxiv2025

AI-Guided Design and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

The structural design of chimeric antigen receptors (CARs) is critical for achieving robust and durable anti-tumor responses, particularly when targeting multiple antigens to prevent tumor antigen escape. However, increasing CAR complexity can introduce structural vulnerabilities, leading to antigen-independent T cell activation, activation-induced cell death, and reduced CAR-T cell persistence. To overcome these challenges, we designed 10,824 CAR molecules across diverse formats and screened 1,452 constructs in-vitro to develop an artificial intelligence model, termed CAR-Mediated Self-Destruction (CARMSeD), which predicts CAR designs susceptible to self-activation and dysfunction. Guided by CARMSeD and structural CAR-CAR interaction modeling, we identified optimized CAR architectures incorporating ICOS and 4-1BB co-stimulatory domains. Humanized bispecific CARs targeting CD20/CD19 and CD22/CD19 demonstrated superior anti-tumor efficacy and persistence both in-vitro and in various xenograft mice models. To further extend CAR-T cell persistence, we engineered bispecific CARs integrated with an AKT3-targeted PROTAC strategy. Targeted degradation of AKT3 enhanced anti-tumor potency, promoted memory T cell formation, and enabled sustained responses even under tumor rechallenge and CD19 antigen-loss conditions. Mechanistically, these effects were mediated by metabolic reprogramming involving FOXO4; notably, FOXO4-deficient CAR-T cells exhibited impaired long-term persistence. Leveraging these mechanistic insights, we developed a trispecific CAR-T cell platform incorporating a bispecific T cell engager (BiTE) targeting CD22/CD3, combined with AKT3 PROTACs. These trispecific CAR-T cells achieved potent tumor eradication, even against malignancies lacking both CD19 and CD20 expression. Collectively, this study presents a comprehensive strategy combining structure-based design, AI-guided screening, and targeted protein degradation to engineer next-generation bi and trispecific CAR-T cells with enhanced persistence, broad antigen coverage, and superior therapeutic durability.

cancer biology↗

bioRxiv2025

Evolutionary trajectories determine the feasibility of collateral sensitivity based antibiotic treatment strategies in critical bacterial pathogens

The rise of antibiotic resistance among pathogenic bacteria necessitates innovative therapeutic strategies. A promising technique is the use of collateral sensitivity where resistance to one antibiotic increases susceptibility to another. In this study, we explored the clinical relevance of collateral sensitivity through experimental evolution and genetic engineering in six critical bacterial pathogens using 23 distinct antibiotics. Our in-depth analysis of Escherichia coli showed that clinically relevant resistance mutations did not confer collateral sensitivity to the tested antibiotics. We were able to identify at least three new classes of ciprofloxacin-resistance mutations that cause collateral sensitivity to multiple antibiotics. However, these mutations incur significant fitness costs and are absent in ciprofloxacin-resistant clinical isolates. Our further analysis showed that the development of collateral effects differed significantly between the tested species. Most species showed development of collateral sensitivity to gentamicin during ciprofloxacin-resistance evolution but Acinetobacter baumanii developed collateral resistance instead. Overall, Pseudomonas aeruginosa showed the most consistent development of collateral sensitivity among the tested species, highlighting it as a promising candidate for the use of collateral-sensitivity-based treatment strategies. Our findings provide insights into the potential of collateral sensitivity as a therapeutic strategy and contribute to the development of more effective antibiotic treatment regimens.

microbiology↗

bioRxiv2024

Behaviorally-relevant features of observed actions dominate cortical representational geometry in natural vision

We effortlessly extract behaviorally relevant information from dynamic visual input in order to understand the actions of others. In the current study, we develop and test different classes of models to better understand the neural representational geometries supporting action understanding. Using fMRI, we measured brain activity as participants viewed a diverse set of 90 different video clips depicting social and nonsocial actions in real-world contexts. We developed five behavioral models using arrangement tasks: two models reflecting behavioral judgments of the purpose (transitivity) and the social content (sociality) of the actions depicted in the video stimuli; and three models reflecting behavioral judgments of the visual content (people, objects, and scene) depicted in still frames of the stimuli. We evaluated how well these models predict neural representational geometry and tested them against semantic models based on verb and nonverb embeddings and visual models based on gaze and motion energy. Our results revealed that behavioral models of action meaning/purpose (transitivity, sociality) better reflect neural representational geometry than behavioral models of object, person, and scene features, as well as semantic and visual models throughout much of cortex. The sociality and transitivity models captured a large portion of unique variance throughout the action observation network, extending into regions not typically associated with action perception, like ventral temporal cortex. Overall, these findings expand our understanding of the action observation network and indicate that the social content and purpose of observed actions are predominant in cortical representation.

neuroscience↗

bioRxiv2023

Transcription attenuation in synthetic promoters in tandem formation

Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in non-overlapping, tandem formation, in single-copy plasmids. From in vivo measurements in E. coli cells, we found that promoters in tandem formation have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which hampers RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to fall-off. The broad spectrum of possible, externally regulated, attenuation strengths in synthetic tandem promoters should make these structures valuable internal regulators of future synthetic circuits.

synthetic biology↗

bioRxiv2023

A library of reporters of the global regulators of gene expression of Escherichia coli

The topology of the transcription factor network (TFN) of E. coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for GFP fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/568972v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@15d7ef4org.highwire.dtl.DTLVardef@139d856org.highwire.dtl.DTLVardef@aa0517org.highwire.dtl.DTLVardef@344a2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Dynamics of bacterial operons during genome-wide stresses

Bacterial gene networks have operons, each coordinating several genes under a primary promoter. Half of the operons in Escherichia coli have been reported to also contain internal promoters. We studied their role during genome-wide stresses targeting key transcription regulators, RNAP and gyrase. Our results suggest that operons responses are influenced by stress-related changes in premature elongation terminations and internal promoters activity. Globally, this causes the responses of genes in the same operon to differ with the distance between them in a wavelike pattern. Meanwhile, premature terminations are affected by positive supercoiling buildup, collisions between elongating and promoter-bound RNAPs, and local regulatory elements. We report similar findings in E. coli under other stresses and in evolutionarily distant bacteria Bacillus subtilis, Corynebacterium glutamicum, and Helicobacter pylori. Our results suggest that the strength, number, and positioning of operons internal promoters might have evolved to compensate for premature terminations, providing distal genes similar response strengths.

systems biology↗

bioRxiv2022

The transcription factor network of E. coli steers global responses to shifts in RNAP concentration

The robustness and sensitivity of gene networks to environmental changes is critical for cell survival. How gene networks produce specific, chronologically ordered responses to genome-wide perturbations, while robustly maintaining homeostasis, remains an open question. We analysed if short- and mid-term genome-wide responses to shifts in RNA polymerase (RNAP) concentration are influenced by the known topology and logic of the transcription factor network (TFN) of Escherichia coli. We found that, at the gene cohort level, the magnitude of the single-gene, mid-term transcriptional responses to changes in RNAP concentration can be explained by the absolute difference between the genes numbers of activating and repressing input transcription factors (TFs). Interestingly, this difference is strongly positively correlated with the number of input TFs of the gene. Meanwhile, short-term responses showed only weak influence from the TFN. Our results suggest that the global topological traits of the TFN of E. coli shape which gene cohorts respond to genome-wide stresses.

systems biology↗

bioRxiv2021

Positive supercoiling buildup is a trigger of E. coli's short-term response to cold shock

Adaptation to cold shock (CS) is a key survival skill of gut bacteria of warm-blooded animals. In E. coli, this skill emerges from a complex transcriptional program of multiple, timely-ordered shifts in gene expression. We identified short-term, cold shock repressed (CSR) genes by RNA-seq and provide evidence that their variability in evolutionary fitness is low and that their responsiveness to cold emanates from intrinsic features. Given that their single-cell variability in protein numbers increases after CS, we hypothesized that the responsiveness of a large portion of CSR genes is triggered by the high propensity for transcription locking due to positive supercoiling buildup (PSB). We then proposed a model of this phenomenon and, in support, show that nearly half of CSR genes are highly responsive to Gyrase inhibition. Also, their response strengths to CS and Gyrase inhibition correlate and most CSR genes increase their single-cell variability in protein numbers. Further, during CS, the cells nucleoid density increases (in agreement with increased numbers of positive supercoils), their energy levels become depleted (while the resolving of positive supercoils is ATP dependent), and the colocalization of Gyrases and the nucleoid increases (in agreement with increased time length for resolving supercoils). We conclude that high sensitivity to PSB is at the core of the short-term, cold shock responsive transcriptional program of E. coli and propose that this gene feature may be useful for providing temperature sensitivity to chromosome-integrated synthetic circuits.

systems biology↗

bioRxiv2021

A general model of the dynamics of genes with dual σ factor preference

Escherichia coli uses {sigma} factors to quickly control large gene cohorts during stress conditions. While most of its genes respond to a single {sigma} factor, approximately 5% of them have dual {sigma} factor preference. The most common are those responsive to both {sigma}70, which controls housekeeping genes, and {sigma}38, which activates genes during stationary growth and stresses. Using RNA-seq and flow-cytometry measurements, we show that {sigma}70+38 genes are nearly as upregulated in stationary growth as {sigma}38 genes. Moreover, we find a clear quantitative relationship between their promoter sequence and their response strength to changes in {sigma}38 levels. We then propose and validate a sequence dependent model of {sigma}70+38 genes, with dual sensitivity to {sigma}38 and {sigma}70, that is applicable in the exponential and stationary growth phases, as well in the transient period in between. We further propose a general model, applicable to other stresses and {sigma} factor combinations. Given this, promoters controlling {sigma}70+38 genes (and variants) could become important building blocks of synthetic circuits with predictable, sequence-dependent sensitivity to transitions between the exponential and stationary growth phases.

systems biology↗

bioRxiv2021

Analytical kinetic model of native tandem promoters in E. coli

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites dTSS, pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS [≤] 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs. Author SummaryTandem promoters are common in nature, but investigations on their dynamics have so far largely relied on synthetic constructs. Thus, their regulation and potentially unique dynamics remain unexplored. We first performed a comprehensive exploration of the conservation of genes regulated by these promoters in E. coli and the properties of their input transcription factors. We then measured protein and RNA levels expressed by 30 Escherichia coli tandem promoters, to establish an analytical model of the expression dynamics of genes controlled by such promoters. We show that start site occlusion and downstream RNAP occupancy can be realistically captured by a model with RNAP binding affinity, the time length of open complex formation, and the nucleotide distance between transcription start sites. This study contributes to a better understanding of the unique dynamics tandem promoters can bring to the dynamics of gene networks and will assist in their use in synthetic genetic circuits.

systems biology↗

bioRxiv2021

Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or {beta}-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by {beta}-lactams. Here we report identification of an allosteric site at a distance of 21 [A] from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second {beta}-lactam molecule and influences binding at the catalytic site. We provide evidence that two {beta}-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-site and one covalently at the catalytic site. This dual {beta}-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new {beta}-lactam antibiotics for M. tuberculosis.

biochemistry↗

bioRxiv2021

The MAP3Ks DLK and LZK direct diverse responses to axon damage in zebrafish peripheral neurons

The MAP3Ks Dual Leucine Kinase (DLK) and Leucine Zipper Kinase (LZK) are essential mediators of axon damage responses, but their responses are varied, complex, and incompletely understood. To characterize their functions in axon injury, we generated zebrafish mutants of each gene, labeled motor neurons (MN) and touch-sensing neurons in live zebrafish, precisely cut their axons with a laser, and assessed the ability of mutant axons to regenerate. DLK and LZK were required redundantly and cell autonomously for axon regeneration in MNs, but not in larval Rohon-Beard (RB) or adult dorsal root ganglion (DRG) sensory neurons. Surprisingly, in dlk lzk double mutants, the spared branches of wounded RB axons grew excessively, suggesting that these kinases inhibit regenerative sprouting in damaged axons. Uninjured trigeminal sensory axons also grew excessively in mutants when neighboring neurons were ablated, indicating that these MAP3Ks are general inhibitors of sensory axon growth. These results demonstrate that zebrafish DLK and LZK promote diverse injury responses, depending on the neuronal cell identity and type of axonal injury. Significance statementThe MAP3Ks DLK and LZK are damage sensors that promote diverse outcomes to neuronal injury, including axon regeneration. Understanding their context-specific functions is a prerequisite to considering these kinases as therapeutic targets. To investigate DLK and LZK cell-type specific functions, we created zebrafish mutants in each gene. Using mosaic cell labeling and precise laser injury we found that both proteins were required for axon regeneration in motor neurons, but, unexpectedly, were not required for axon regeneration in Rohon-Beard or dorsal root ganglion (DRG) sensory neurons, and negatively regulated sprouting in the spared axons of touch-sensing neurons. These findings emphasize that animals have evolved distinct mechanisms to regulate injury site regeneration and collateral sprouting, and identify differential roles for DLK and LZK in these processes.

neuroscience↗

bioRxiv2021

Plasmodium falciparum and helminth coinfections increase IgE and parasite-specific IgG responses

Coinfection with Plasmodium falciparum and helminths may impact the immune response to these parasites since they induce different immune profiles. We studied the effects of coinfections on the antibody profile in a cohort of 715 Mozambican children and adults using the Luminex technology with a panel of 16 antigens from P. falciparum and 11 antigens from helminths (Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis and Schistosoma spp.) and measured antigen-specific IgG and total IgE responses. We compared the antibody profile between groups defined by P. falciparum and helminth previous exposure (based on serology) and/or current infection (determined by microscopy and/or qPCR). In multivariable regression models adjusted by demographic, socioeconomic, water and sanitation variables, individuals exposed/infected with P. falciparum and helminths had significantly higher total IgE and antigen-specific IgG levels, magnitude (sum of all levels) and breadth of response to both types of parasites compared to individuals exposed/infected with only one type of parasite (p[≤] 0.05). There was a positive association between exposure/infection to P. falciparum and exposure/infection to helminths or the number of helminth species, and vice versa (p[≤] 0.001). In addition, children coexposed/coinfected tended (p= 0.062) to have higher P. falciparum parasitemia than those single exposed/infected. Our results suggest that an increase in the antibody responses in coexposed/coinfected individuals may reflect higher exposure and be due to a more permissive immune environment to infection in the host. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/445753v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@a1dc3corg.highwire.dtl.DTLVardef@82128corg.highwire.dtl.DTLVardef@18fcd98org.highwire.dtl.DTLVardef@2d5bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Fluorescent Nanosensors Reveal Dynamic pH Gradients During Biofilm Formation

Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities. The components and architecture of biofilms have been interrogated in depth, however, little is known about the environmental microniches present. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a novel tool, to observe physiological pH changes in biofilms in real-time. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. The nanosensors were used to analyse the real-time three-dimensional pH variation for two model biofilm formers: (i) opportunistic pathogen Pseudomonas aeruginosa, and (ii) oral pathogen Streptococcus mutans. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify novel therapeutic solutions to improve oral health.

microbiology↗