Biology subjects

deMello, A.

Publications and source records attributed to deMello, A..

12 recordsLinked to original sources

bioRxiv2026

Structural mechanism governing radiationless energy transfer in Renilla bioluminescence

The nonradiative transport of electronic excitation from one chromophore to another, known as resonance energy transfer, lies at the root of photochemical processes in biology. Unlike photosynthesis, bioluminescence converts chemical energy into light through an enzymatic oxygenation of an energy-rich luciferin. In glowing cnidarians, the energy is relocated from an excited oxyluciferin to a fluorescent protein, shifting the colour and enhancing the quantum yield of a photogenic reaction. How protein-chromophore complexes assemble during this interplay in real space, and what this association entails for function, are unknown. Here, we report co-crystal structures of a 120-kilodalton energy-transfer complex from the luminescent soft coral Renilla reniformis. We find a heterotetrameric 2:2 assembly composed of two coelenteramide-loaded luciferases (RrLuc) docked at opposite sides of a head-to-tail dimer of green fluorescent protein (RrGFP). The edge-to-edge distance between donor and acceptor chromophores is below 3 nm, favouring the Forster-type radiationless energy transfer. Furthermore, RrGFP serves not only as a colour-switchable antenna and luminescence amplifier but also tunes the efficiency of luciferase catalysis by controlling its inherent dynamics. Our results provide detailed spatial information about intermolecular dipole-dipole coupling in Renilla bioluminescence, including the arrangement of donor-acceptor pairs that secure excited-state energy transfer with exquisite precision.

biochemistry↗

bioRxiv2026

Droplet single-cell CRISPR screens identify regulators of T cell-mediated target-cell killing

Cytotoxic CD8 T cells kill target cells through brief cell-cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8 T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN, RASA2 and FOXO1, together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.

immunology↗

bioRxiv2026

CascadeMAP: Autonomous Closed-loop Optimization of Enzyme Cascades via Microfluidics, Machine Learning and Agentic AI

Enzyme cascades enable complex biochemical transformations, but their optimization is resource-intensive, requiring navigation through high-dimensional parameter spaces encompassing reaction conditions, enzyme ratios, and buffer composition. Here we introduce CascadeMAP, an autonomous microfluidic platform for closed-loop optimization of enzyme cascades, integrating high-throughput microfluidics with Bayesian optimization and multi-agent AI system. We demonstrate the platform across two cascades: (i) a glycerol detection pathway monitored by fluorescence and (ii) a 1,2,3-trichloropropane degradation pathway monitored by label-free Raman spectroscopy providing orthogonal detection modalities. Bayesian optimization identified optimal conditions three times faster than Design of Experiments. Multi-agent AI system automated hypothesis generation, processing 11 GB of experimental data, pattern recognition, and insight synthesis. Operating without human intervention for 7 days, CascadeMAP processed [~]220,000 reactions across [~]7,400 different conditions. This capability establishes a generalizable framework for the autonomous optimization of enzyme cascades and metabolic pathways and accelerates the development of biocatalytic and synthetic biological systems.

biochemistry↗

bioRxiv2026

Uncovering Functional Distant Mutations by Ultra-High-Throughput Screening of Dehalogenases

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Kinetic mechanism of Renilla luciferase guides induced-fit engineering for improved bioluminescence

Luciferases are widely used bioluminescent reporters, yet the molecular determinants of their catalytic efficiency and light-emission stability remain incompletely understood. Here, we reconstruct the complete catalytic pathway of Renilla luciferase by combining steady-state, transient, and temperature-dependent kinetics with crystallography and molecular simulations. We show that the enzyme is substantially undersaturated with oxygen (Km,O2 = 719 M), causing its true turnover number (kcat = 21.9 s-1) to be systematically underestimated. Concurrently, elevated oxygen drives irreversible enzyme inactivation after ~1,500 turnovers, revealing a fundamental trade-off that limits oxygen-affinity engineering. Instead, the genuine bottleneck of the catalytic cycle is the induced-fit conformational opening of the product-bound enzyme. Selective engineering of this transition step through rational loop grafting yielded a variant AncFT-L14 with enhanced catalytic efficiency and glow-type bioluminescence with substantially slower signal decay in cell lysates. Collectively, our results identify conformational dynamics as a primary tunable determinant of luciferase function. More broadly, this work establishes a mechanistically grounded framework for the development of next-generation bioluminescent tools and for engineering enzymes controlled by dynamically gated ligand exchange.

biochemistry↗

bioRxiv2023

Viscoelastic Deformability Cytometry: Ultra-high Throughput Platform for Mechanical Phenotyping of Cells in Liquid and Solid Biopsies

Due to the differences in mechanical properties between cancer cells and their benign counterparts, the mechanical characteristics of cells have emerged as a significant feature in diagnosing cancer. Consequently, measuring cell deformability as a means of mechanical phenotyping is promising for both the detection and classification of the disease. However, performing high-throughput single-cell deformability measurements on liquid or solid tissue biopsies remains a significant challenge within a clinical setting. Herein, we introduce an ultra-high throughput viscoelastic-based microfluidic platform to measure the cell mechanical properties at rates of up to 100,000 cells/s. Thanks to the viscoelastic property of the fluid, cells are focused and deformed within the same platform obviating the need for any sheath fluid. We used the presented platform for cell phenotyping in both liquid and solid tumor biopsies, such as identification of malignant lymphocytes in peripheral blood samples and glioblastoma-type cell classification from solid tumor samples. The presented platform has the potential to open new opportunities in the assessment of cancer, enable precise mechanical profiling of rare cells, and facilitate sensitive diagnostic applications.

bioengineering↗

bioRxiv2023

Actomyosin-mediated apical constriction promotes physiological germ cell death in C. elegans

Germ cell apoptosis in C. elegans hermaphrodites is a physiological process eliminating around 60% of all cells in meiotic prophase to maintain tissue homeostasis. In contrast to programmed cell death in the C. elegans soma, the selection of germ cells undergoing apoptosis is stochastic. By live-tracking individual germ cells at the pachytene stage, we found that germ cells smaller than their neighbors are selectively eliminated through apoptosis before differentiating into oocytes. Thus, cell size is a strong predictor of physiological germ cell death. The RAS/MAPK and ECT/RHO/ROCK pathways together regulate germ cell size by controlling actomyosin constriction at the apical rachis bridges, which are cellular openings connecting the syncytial germ cells to a shared cytoplasmic core. Enhancing apical constriction reduces germ cell size and increases the rate of cell death while inhibiting the actomyosin network in the germ cells prevents their death. We propose that actomyosin contractility at the rachis bridges of the syncytial germ cells amplifies intrinsic disparities in cell size. Through this mechanism, animals can adapt the rate of germ cell death and differentiation to changing environmental conditions.

developmental biology↗

bioRxiv2021

Microfluidic-based imaging of complete C. elegans larval development

Several microfluidic-based methods for long-term C. elegans imaging have been introduced in recent years, allowing real-time observation of previously inaccessible processes. The existing methods either permit imaging across multiple larval stages without maintaining a stable worm orientation, or allow for very good immobilization but are only suitable for shorter experiments. Here, we present a novel microfluidic imaging method, which allows parallel live-imaging across multiple larval stages, while delivering excellent immobilization and maintaining worm orientation and identity over time. This is achieved by employing an array of microfluidic trap channels carefully tuned to maintain worms in a stable orientation, while allowing growth and molting to occur. Immobilization is supported by an active hydraulic valve, which presses worms onto the cover glass during image acquisition, with the animals remaining free for most of an experiment. Excellent quality images can be acquired of multiple worms in parallel, with little impact of the imaging method on worm viability or developmental timing. The capabilities of this methodology are demonstrated by observing the hypodermal seam cell divisions and, for the first time, the entire process of vulval development from induction to the end of morphogenesis. Moreover, we demonstrate RNAi on-chip, which allows for perturbation of dynamic developmental processes, such as basement membrane breaching during anchor cell invasion.

developmental biology↗

bioRxiv2020

In-vitro and in-vivo characterization of CRANAD-2 for multi-spectral optoacoustic tomography and fluorescence imaging of amyloid-beta deposits in Alzheimer mice

The abnormal deposition of fibrillar beta-amyloid (A{beta}) deposits in the brain is one of the major histopathological hallmarks of Alzheimers disease (AD). Here we characterize curcumin-derivative CRANAD-2 for multi-spectral optoacoustic tomography (MSOT) and fluorescence imaging of brain A{beta} deposits in the arcA{beta} mouse model of AD cerebral amyloidosis. CRANAD-2 shows a specific and quantitative detection of A fibrils in vitro, even in complex mixtures, and it is capable to distinguish between monomeric and fibrillar forms of A. In vivo epifluorescence and MSOT after intravenous CRANAD-2 administration demonstrated higher retention in arcA{beta} compared to non-transgenic littermate mice. Immunohistochemistry showed co-localization of CRANAD-2 and A{beta} deposits in arcA{beta} mouse brain sections, thus verifying the specificity of the probe. In conclusion, we demonstrate suitability of CRANAD-2 for fluorescence- and MSOT-based detection of A{beta} deposits in animal models of AD pathology, which facilitates mechanistic studies and the monitoring of putative treatments targeting A{beta} deposits.

neuroscience↗

bioRxiv2019

Heterogeneity in heat shock response dynamics caused by translation fidelity decline and proteostasis collapse

Genetics, environment, and stochasticity influence the rate of ageing in living organisms. Individual Caenorhabditis elegans that are genetically identical and cultured in the same environment have different lifespans, suggesting a significant role of stochasticity in ageing. We have developed a novel microfluidic methodology to measure heat-shock response as a surrogate marker for heterogeneity associated with lifespan and have quantified the heat-shock response of C. elegans at the population, single individual, and tissue levels. We have further mathematically modelled our data to identify the major drivers determining such heterogeneity. This approach demonstrates that protein translation and degradation rate constants explain the individuality of the heat-shock time-course dynamic. We observed a decline of protein turnover capacity in early adulthood, co-incidentally occurring as the predicted proteostasis collapse. We identified a decline of intestinal response as the tissue that underlies the individual heterogeneity. Additionally, we verified that individuals with enhanced translation fidelity in early adulthood live longer. Altogether, our results reveal that the stochastic onset of proteostasis collapse of somatic tissues during early adulthood reflects individual protein translation capacity underlying heterogenic ageing of isogenic C. elegans.

bioengineering↗

bioRxiv2019

Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection

Flow cytometry is widely recognized as the gold-standard technique for the analysis and enumeration of heterogeneous cellular populations and has become an indispensable tool in diagnostics,1 rare-cell detection2 and single-cell proteomics.3 Although contemporary flow cytometers are able to analyse many thousands of cells per second, with classification based on scattering or fluorescence criteria, the vast majority require unacceptably large sample volumes, and do not allow the acquisition of spatial information. Herein, we report a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence and brightfield detection at analytical throughputs in excess of 60,000 cells/s and 400,000 cells per second respectively. Our imaging platform is capable of multi-parametric fluorescence quantification and subcellular (co-)localization analysis of cellular structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of our approach by performing challenging high-throughput localization analysis of cytoplasmic RNA granules in yeast and human cells. Results suggest significant utility of the imaging flow cytometer in the screening of rare events at the subcellular level for diagnostic applications.

bioengineering↗

bioRxiv2019

Oscillatory Viscoelastic Microfluidics for Efficient Focusing and Separation of Nanoscale Species

The ability to precisely control particle migration within microfluidic systems is essential for focusing, separating, counting and detecting a wide range of biological species. To date, viscoelastic microfluidic systems have primarily been applied to the focusing, separation and isolation of micron-sized species, with their use in nanoparticle manipulations being underdeveloped and underexplored, due to issues related to nanoparticle diffusivity and a need for extended channel lengths. To overcome such issues, we herein present sheathless oscillatory viscoelastic microfluidics as a method for focusing and separating both micron and sub-micron species. To highlight the efficacy of our approach, we segment our study into three size regimes, namely micron (where characteristic particle dimensions are above 1 m), sub-micron (where characteristic dimensions are between 1 m and 100 nm) and nano (where characteristic dimensions are below 100 nm) regimes. Based on the ability to successfully manipulate particles in all these regimes, we demonstrate the successful isolation of p-bodies from biofluids (in the micron regime), the focusing of {lambda}-DNA (in the sub-micron regime) and the focusing of extracellular vesicles (in the nano-regime). Finally, we characterize the physics underlying viscoelastic microflows using a dimensionless number that relates the lateral velocity (due to elastic effects) to the diffusion constant of the species within the viscoelastic carrier fluid. Based on the ability to precisely manipulate species in all three regimes, we expect that sheathless oscillatory viscoelastic microfluidics will provide for significant new opportunities in a range of biological and life science applications.

bioengineering↗