Enzymatic Cascade Research Articles

Near-infrared fluorescent probe based on carboxylesterase 2 and viscosity cascade response for early diagnosis of thyroid cancer

Carboxylesterase 2 (CES2) plays a critical role in the metabolism of ester- and amide-containing drugs and thus has an impact on drug efficacy and toxicity. This study introduces QVJ-CES2, a near-infrared fluorescent probe designed to enhance thyroid cancer diagnosis through CES2 enzyme activity and distinct cellular viscosity of thyroid cells. The probe's dual-response mechanism significantly amplified fluorescence in high-viscosity environments, thus had high sensitivity. Our findings revealed that QVJ-CES2 could detect CES2 activity with a limit of detection as low as 1.9 ng/mL. In vitro experiments confirmed the rapid and selective response of QVJ-CES2 to CES2 and the ability to distinguish cancer cells from their normal cells with high specificity. Most importantly, the fluorescence intensity difference between cancerous and normal cells after incubation with the probe was as high as 7-fold, attributable to the synergistic enzyme and viscosity cascade response. Further, in vivo studies demonstrated that the probe was effective in accurately identifying thyroid cancer tissues, showing that it is a promising tool for early cancer detection and precise surgical interventions.

Counter-regulatory RAS peptides: new therapy targets for inflammation and fibrotic diseases?

The renin-angiotensin system (RAS) is an important cascade of enzymes and peptides that regulates blood pressure, volume, and electrolytes. Within this complex system of reactions, its counter-regulatory axis has attracted attention, which has been associated with the pathophysiology of inflammatory and fibrotic diseases. This review article analyzes the impact of different components of the counter-regulatory axis of the RAS on different pathologies. Of these peptides, Angiotensin-(1-7), angiotensin-(1-9) and alamandine have been evaluated in a wide variety of in vitro and in vivo studies, where not only they counteract the actions of the classical axis, but also exhibit independent anti-inflammatory and fibrotic actions when binding to specific receptors, mainly in heart, kidney, and lung. Other functional peptides are also addressed, which despite no reports associated with inflammation and fibrosis to date were found, they could represent a potential target of study. Furthermore, the association of agonists of the counter-regulatory axis is analyzed, highlighting their contribution to the modulation of the inflammatory response counteracting the development of fibrotic events. This article shows an overview of the importance of the RAS in the resolution of inflammatory and fibrotic diseases, offering an understanding of the individual components as potential treatments.

Design of a synthetic enzyme cascade for the in vitro fixation of formaldehyde to acetoin

Formaldehyde (FALD) has gained prominence as an essential C1 building block in the synthesis of valuable chemicals. However, there are still challenges in converting FALD into commodities. Recently, cell-free biocatalysis has emerged as a popular approach for producing such commodities. Acetoin, also known as 3-hydroxy-2-butanone, has been widely used in food, cosmetic, agricultural and the chemical industry. It is valuable to develop a process to produce acetoin from FALD. In this study, a cell-free multi-enzyme catalytic system for the production of acetoin using FALD as the substrate was designed and constructed. It included three scales: FALD utilization pathway, glycolysis pathway and acetoin synthesis pathway. After the optimization of the reaction system, 20.17 mM acetoin was produced from 122 mM FALD, with a yield of 0.165 mol/mol, reaching 99.0% of the theoretical yield. The pathway provides a new approach for high-yield acetoin production from FALD, which consolidates the foundation for the production of high value-added chemicals using cheap one-carbon compounds.

Distance-based paper sensor for DNase I detection based on aptamer-mediated enzyme cascade reaction

DNase I is proven to be an early predictive molecule for a variety of diseases, which is important for early diagnosis and clinical detection of diseases. Herein, we demonstrate a distance-based paper sensor to detect DNase I via an enzymatic cascade reaction mediated by the thrombin aptamer. In the presence of DNase I, the thrombin is able to clot the plasma normally because the aptamer chain is hydrolyzed by DNase I and cannot bind thrombin properly to inhibit thrombin activity. The concentration of DNase I is negatively correlated with the volume of mobile phase in sample solution, and it can be quantified by measuring the flow distance of water on the pH indicator strip. The distance-based paper sensor has good specificity, and the detection limit is 2 × 10−2 U/mL. In addition, it can be used for DNase I analysis in human serum samples to show its efficacy in the early diagnosis of diseases associated with DNase I activity. Overall, we developed a simple, fast and low-cost DNase I detection method, which has great commercial potential.

Injectable Hydrogel Based on Enzymatic Initiation of Keratin Methacrylate for Controlled Exosome Release in Intervertebral Disc Degeneration Therapy

AbstractThe treatment of intervertebral disc degeneration (IVDD) using bone marrow mesenchymal stem cell‐derived exosomes has shown success in alleviating inflammation and restoring the extracellular matrix (ECM), however, challenges persist due to the deficiency in mechanical support and controlled release. Herein, a carbon‐carbon double bond modified keratin (KeMA) is synthesized by 2‐isocyanatoethyl modification for exosomes wrapping. This injectable KeMA hydrogel, initiated by a biocompatible glucose/ glucose oxidase/ horse radish peroxidase enzymatic cascade reaction with acetylacetone and N‐vinylpyrrolidone, displayed rapid gelation, resembling nucleus pulposus (NP) elasticity, and excellent cytocompatibility. In vitro studies showcased that the exosomes‐loaded KeMA hydrogel (Exo@KeMA) enhanced exosome release kinetics, suppressed inflammation, fostered extracellular matrix (ECM) regeneration, and reinstated NP biomechanics. RNA‐seq analysis indicated Exo@KeMA's effects involved PI3K‐Akt signaling for matrix regeneration and NF‐κB signaling inhibition for anti‐inflammation. In vivo IVDD rat models demonstrated Exo@KeMA attenuated inflammation, maintained NP water content, preserved disc height, and promoted structural regeneration. This research introduces an injectable KeMA hydrogel as a promising therapy for IVDD, by facilitating biomechanics restoration, anti‐inflammatory response, and ECM regeneration.

Mechanically operated signalling scaffolds.

Cellular signalling is a complex process and involves cascades of enzymes that, in response to a specific signal, give rise to exact cellular responses. Signalling scaffold proteins organise components of these signalling pathways in space and time to co-ordinate signalling outputs. In this review we introduce a new class of mechanically operated signalling scaffolds that are built into the cytoskeletal architecture of the cell. These proteins contain force-dependent binary switch domains that integrate chemical and mechanical signals to introduce quantised positional changes to ligands and persistent alterations in cytoskeletal architecture providing mechanomemory capabilities. We focus on the concept of spatial organisation, and how the cell organises signalling molecules at the plasma membrane in response to specific signals to create order and distinct signalling outputs. The dynamic positioning of molecules using binary switches adds an additional layer of complexity to the idea of scaffolding. The switches can spatiotemporally organise enzymes and substrates dynamically, with the introduction of ∼50 nm quantised steps in distance between them as the switch patterns change. Together these different types of signalling scaffolds and the proteins engaging them, provide a way for an ordering of molecules that extends beyond current views of the cell.

Towards Autonomous Materials–Challenges in Chemical Communication

AbstractCoordination of functions in multi‐body or multi‐component systems requires communication among its parts and is a prerequisite for achieving complex tasks. While electromagnetic (network) communication provides a key ingredient for modern robotics, its molecular equivalent is largely missing for soft robots. With advancements in programmed cargo release, DNA strand‐displacement reactions, enzymatic cascades, and genetic circuits, budding solutions to such chemical network communication have emerged with the potential to drive function in soft machines. In order for such chemical communication to be useful, however, new control concepts, (orthogonal) communication protocols, and molecular solutions should be found. Herein, we provide a critical perspective on the current state‐of‐the‐art chemical communication and identify key challenges towards autonomous soft materials, including signal processing, dealing with noise, switching signaling modalities, and limitations in time and length scales that determine material design. Building on an emerging body of examples, we illustrate gaps, synergies, and new concepts that may provide possible solutions to achieve standardized and reliable molecular information exchange for regulating (soft) robotic function.

Genistein effect in cultured ovine ovarian granulosa cells.

Genistein, an isoflavone has the potential to mimic, augment, or dysregulate the steroid hormone production pathways. We hypothesized that genistein affects the granulosa cell (GCs) functions through a series of biochemical, molecular, and genomic cascades. The present study was conducted to evaluate the impact of genistein exposure on GCs viability, apoptosis, and steroidogenesis. The present study involved 3/5 days of exposure to genistein on GCs collected from abattoir-derived ovine ovaries at doses of 0, 1, 10, 25, 50, and 100 µM. The harvested GCs were used for growth, cytotoxicity, and gene expression studies related to apoptosis, growth, and steroidogenesis. We observed that genistein had both stimulatory at 10 and 25 µM levels as well as inhibitory effects at 50 and 100 µM levels on the growth and proliferation of GCs. Genistein significantly decreased the levels of 17β-estradiol at higher exposure (50 and 100 µM), whereas the progesterone level increased significantly as the genistein exposure increased. Additionally, genistein could also alter the mRNA expression of the steroidogenic receptor, enzymes, proteins, and growth-related genes suggesting that genistein could potentially alter the steroidogenic pathways. We conclude that genistein can interfere with cell survival and steroidogenesis by exhibiting a dose-dependent biphasic response on the viability, growth-related parameters, and the synthesis of 17β-estradiol in the cultured GCs.

Psychiatric disorder in prolonged post-concussive syndrom : clinical assessment, physiopathology and management review of the literature

IntroductionAccording to the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), post concussive syndrome (PCS) is given a diagnosis of either major or mild neurocognitive disorder (NCD) due to traumatic brain injury TBI. However Persistent post-concussion symptoms (PPCS) are more complex, and typically involve multidisciplinary assessment and management. The symptoms are varied, non-specific and the therapeutic process is defiant for psychiatrist.ObjectivesTo investigate the semiology of persistent post-concussion syndrome (PPCS) and the therapeutic challenges it poses.MethodsA literature review was made on Pubmed, Google Scholar and Cochrane library using keywords: “post-concussive syndrome”,”psychiatric disorder”, “depression”,”post-traumatic stress disorder”,”treatment”, “physiopathology”.ResultsThe physiopathology of persistent PCS is controversy.The Symptoms are due to the Concept of “Symptom Generators” which results from the alterations in neurophysiology and neuropathology secondary to the injury, and pre- or post-injury psychological factors physiological concussion. The Global cerebral metabolic disturbance, the autonomic nervous system dysfunction and the cerebral blood flow dysregulation induce biochemical cascade,excitotoxic reaction and immunotoxicity.Clinical diagnoses associated with PPCS are:Major depressive disorder,Post traumatic stress disorder,Anxiety disorder,Substance abuse disorder,Psychotic disorder and Antisocial personality disorder. For the non pharmacological management: A systematically early information and a graded physical exercise in addition to other treatment are essential.Antidepressant, benzodiazepine and mood-stabilizer are the most recommended treatments for psychiatric symptoms. Atypical neuroleptics are indicated in delirant disorder, behavior disorder and antisocial personality disorder.Some studies suggest the methylphenidate and biperiden to treat several cognitive impairment and severe behavior disorder.Conclusions(PPCS) is far from being a subjective complaint by patients. It is a complex clinical entity that groups symptoms that overlap with other psychiatric diagnoses, such as depression, post-traumatic stress disorder, and mood disorders. Early neuropsychiatric assessment and personalized pharmacological and psychotherapeutic treatment are essential factors in the prognosis of the disease.Disclosure of InterestNone Declared

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyses the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one (SDO) to a conjugated (22, 24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an FMN cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by SDO but cannot be oxidized even with strong oxidising agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2 (S26DH2). In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Discovery and characterization of noncanonical E2-conjugating enzymes.

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

Design, synthesis, antimicrobial evaluation, and molecular modeling of new sulfamethoxazole and trimethoprim analogs as potential DHPS/DHFR inhibitors

Trimethoprim (TMP) and sulfonamides and are typically utilized to hinder the activities of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively. In this context, we aimed to enhance the understanding of the inhibitory effects on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes by synthesizing and evaluating two series of compounds. These compounds, designed as derivatives of p-aminobenzoic acid (related to sulfamethoxazole, SUL) and trimethoprim analogs, were rigorously tested for their in vitro antimicrobial efficacy and their ability to hinder the activities of DHFR and DHPS enzymes.Among the synthesized compounds, compounds 6 and 10 demonstrated exceptional antimicrobial efficacy against a diverse spectrum of bacterial and fungal strains. The assessment of these compounds also included their impact on biofilm formation, evaluated through crystal violet staining. Our in vitro enzyme assays revealed that compound 10 emerged as the most potent inhibitor of DHPS, with an IC50 value of 224.91 μg/mL, surpassing SUL by 1.6 times. Simultaneously, Compound 6 exhibited particularly potent DHFR-inhibitory action, with an IC50 value of 3.731 μg/mL, surpassing TMP by a factor of three.Molecular modeling studies further elucidated the mode of action, illustrating that compound 10 effectively occupied the DHPS pterin-binding site alongside p-aminobenzoic acid. In contrast, compound 6 was adeptly positioned within the DHFR-binding pocket.In summary, our findings underscore the promising prospects of compounds 6 and 10 in the management of bacterial infections. The results not only demonstrate their remarkable antimicrobial efficacy but also position them as potentially effective in a synergistic combination targeting the DHPS/DHFR enzymatic cascade.

A novel NiO/CeO2-mSiO2 nanocages with peroxidase-mimetic activity for ultra-sensitive biosensing

A novel NiO/CeO2−mSiO2 nanocages with superior peroxidase−like activities due to the synergetic effect between NiO and CeO2 were fabricated by a simple etching−precipitation method with core−shell structured Cu2O@mSiO2 as a sacrificial template. Based on the peroxidase−like activity of the NiO/CeO2−mSiO2 nanocages, an enzyme cascade−triggered colorimetric sensing platform was exploited for alkaline phosphatase (ALP) detection, owing to the fact that ascorbic acid (AA) that is the ALP enzymatic hydrolysis product of ascorbic acid 2−phosphate (AAP) can efficiently reduce the oxidation product of TMB (oxTMB) generated under the catalysis of the NiO/CeO2−mSiO2. For the sensing of ALP, a good linear response was observed in the range of 0.0005–1.25 mU mL−1 with the limit of detection (LOD) as low as 0.00048 mU mL−1. Furthermore, the proposed enzyme cascade−triggered colorimetric sensing platform could be further explored for sensing of tyrosinase, according to the fact that dopamine (DA) the catalytic product of tyramine (Tyr) by tyrosinase could initiate the redox reaction with the oxTMB acquired under the catalysis of the NiO/CeO2−mSiO2. A wide linear response was found for tyrosinase detection from 0.02 mU mL−1 to 3.4 U mL−1 with a LOD as low as 0.002 U mL−1. This work not only offers a facile and effective approach to fabricate nanozymes with excellent catalytic activity, but also provides a promising enzyme cascade−triggered colorimetric method for the ultra−sensitive detection of biological enzymes.

Whole-cell catalytic production of pseudouridine by recombinant Escherichia coli

Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.

Double-Network structure chemically programmed DNA Cryogel: Enzyme immobilization platform with enhanced catalytic activity for portable biosensing

Enzyme-based hydrogels have conferred multiple benefits, particularly for disease diagnosis and environmental monitoring due to their pronounced biocompatibility, ability to load cargo, and optical properties. However, traditional hydrogels suffer from some significant limitations, such as enzyme leakage and catalytic activity decreased, restricting the extent to which hydrogels can be beneficially used. Herein, chemically programmed DNA-DNA interactions were designed as a promising solution for enzyme immobilization that substantially maintains biological functionality with remarkable leakage reduction. Additionally, their responsiveness and mechanical properties are dramatically enhanced after adjusting the porous structure of hydrogels via freezing. As a proof-of-concept, a DNA cryogel with a hierarchical structure was developed with adjustable distance among cascade enzymes, increasing the local concentration of intermediates and reducing their mass transfer energy barriers for direct glucose monitoring and glutathione (GSH) sensing. Moreover, after being integrated with a smartphone system, a universal sensing platform (immunosensor) was fabricated for rapid detection of environmental chemicals (i.e., bisphenol A) without expensive instruments and sophisticated procedures. This strategy provided some novel insights on hydrogel- based bioassays, implying their great potentials for conducting onsite analysis against various targets.

Semiconducting polymer dots based l-lactate sensor by enzymatic cascade reaction system

Backgroundl-lactate detection is important for not only assessing exercise intensity, optimizing training regimens, and identifying the lactate threshold in athletes, but also for diagnosing conditions like L-lactateosis, monitoring tissue hypoxia, and guiding critical care decisions. Moreover, l-lactate has been utilized as a biomarker to represent the state of human health. However, the sensitivity of the present l-lactate detection technique is inadequate. ResultsHere, we reported a sensitive ratiometric fluorescent probe for l-lactate detection based on platinum octaethylporphyrin (PtOEP) doped semiconducting polymer dots (Pdots-Pt) with enzymatic cascade reaction. With the help of an enzyme cascade reaction, the l-lactate was continuously oxidized to pyruvic and then reduced back to l-lactate for the next cycle. During this process, oxygen and NADH were continuously consumed, which increased the red fluorescence of Pdots-Pt that responded to the changes of oxygen concentration and decreased the blue fluorescence of NADH at the same time. By comparing the fluorescence intensities at these two different wavelengths, the concentration of l-lactate was accurately measured. With the optimal conditions, the probes showed two linear detection ranges from 0.5 nM to 5.0 μM and 5.0 μM–50.0 μM for l-lactate detection. The limit of detection was calculated to be 0.18 nM by 3σ/slope method. Finally, the method shows good detection performance of l-lactate in both bovine serum and artificial serum samples, indicating its potential usage for the selective analysis of l-lactate for health monitoring and disease diagnosis. SignificanceThe successful application of the sensing system in the complex biological sample (bovine serum and artificial serum samples) demonstrated that this method could be used for sensitive l-lactate detection in practical clinical applications. This detection system provided an extremely low detection limit, which was several orders of magnitude lower than methods proposed in other literatures.

Encapsulation of Enzymes into Hydrophilic and Biocompatible Metal Azolate Framework: Improved Functions of Biocatalyst in Cascade Reactions and its Sensing Applications.

Multiple enzyme-triggered cascade biocatalytic reactions are vital in vivo or vitro, considering the basic biofunction preservation in living organisms and signals transduction for biosensing platforms. Encapsulation of such enzymes into carrier endows a sheltering effect and can boost catalytic performance, although the selection and preparation of an appropriate carrier is still a concern. Herein, focusing on MAF-7, a category of metal azolate framework (MAF) with superiority against the topologically identical ZIF-8, this enzyme@MAF system can ameliorate the sustainability of encapsulating natural enzymes into carriers. The proposed biocatalyst composite AChE@ChOx@MAF-7/hemin is constructed via one-pot in situ coprecipitation method. Subsequently, MAF-7 is demonstrated to exhibit an excellent capacity of the carrier and protection against external factors in the counterpart of ZIF-8 through encapsulated and free enzymes. In addition, detections for specific substrates or inhibitors with favorable sensitivity are accomplished, indicating that the properties above expectation of different aspects of the established platform are successfully realized. This biofunctional composite based on MAF-7 can definitely provide a potential approach for optimization of cascade reaction and enzyme encapsulation.

Abstract 3596: Metabolic reprogramming of the tumor microenvironment to enhance the functionality of TCR-edited T cell in colorectal cancer and liver metastases

Abstract In solid malignancies, T cells functionality is intertwined with metabolic reprogramming within the tumor microenvironment (TME), which supports the increased energy request of cancer cells and suppresses the effector function and cytokines production of the immune infiltrate. In primary colorectal cancer (CRCs) and related liver metastases, we revealed extensive transcriptional remodeling across tumors, being metabolic pathways among the major drivers of this variance. Accordingly, extracellular adenosine, accumulating in the TME for the hydrolysis of ATP by CD39 and CD73, has been recognized as a novel inhibitory mediator, which binds preferentially to the A2A receptor (A2Ar) to exert its immunosuppressive activity. By high dimensional flow cytometry, we revealed that CD39 is a major driver of T-cell exhaustion in primary and metastatic CRCs. Therefore, we sought to disrupt CD39 in TCR-edited T cells (TCRED). By CRISPR/Cas9, we disrupted both the α and β chains of the endogenous TCR together with ENTPD1, the gene encoding for CD39, and redirected T cell specificity against the HER2 antigen. TCREDCD39KO T cells outperformed their competent counterpart in eliminating patient-derived organoids (PDOs) from primary and metastatic tumors both in vitro and in vivo. Given the role of CD39 in starting the biochemical cascade leading to adenosine production, we aimed at investigating the direct role of adenosine on the functional advantage observed with TCREDCD39KO T cells. We challenged our cellular products with target cells in the presence or absence of adenosine and observed that TCREDCD39KO T cells are superior to their competent counterpart in the ability to release cytokines, while the effect is completely abolished when adenosine is present. Also, when TCREDCD39KO T cells were challenged with PDOs, the exogenously added adenosine nullifies the functional killing advantage given by the CD39 KO. To gain more insights into the pathway, we also generated TCREDA2ArKO T cells. In line with the different roles of CD39 and A2AR on the ATP/adenosine pathway, the disruption of the adenosine receptor resulted in a significant advantage in PDO killing in the presence of adenosine only. We then evaluated the influence of CD39KO in the metabolic capacity of T cells and revealed that TCREDCD39KO T cells have a greater potential to rely on the mitochondrial other than glycolytic functions, which explains higher functionality and persistence. Further evaluation on the metabolic effect of CD39 KO are currently ongoing. Overall, we showed that the ATP/adenosine cascade is relevant for T cells functionality and metabolic status and can be harnessed in adoptive cell therapy strategies to counteract the immunosuppressive TME. Citation Format: Alessia Potenza, Chiara Balestrieri, Martina Spiga, Luca Albarello, Federica Pedica, Arianna Ferrari, Oronza A. Botrugno, Barbara Camisa, Elena Tiziano, Camilla Sirini, Monica Casucci, Chiara Iozzi, Danilo Abbati, Ugo Elmore, Giulia Di Lullo, Giulia Casorati, Claudio Doglioni, Giovanni Tonon, Paolo Dellabona, Riccardo Rosati, Luca Aldrighetti, Paolo Monti, Eliana Ruggiero, Chiara Bonini. Metabolic reprogramming of the tumor microenvironment to enhance the functionality of TCR-edited T cell in colorectal cancer and liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3596.

Mechanosensitive TRPV4 channel guides maturation and organization of the bilayered mammary epithelium

Biophysical cues from the cell microenvironment are detected by mechanosensitive components at the cell surface. Such machineries convert physical information into biochemical signaling cascades within cells, subsequently leading to various cellular responses in a stimulus-dependent manner. At the surface of extracellular environment and cell cytoplasm exist several ion channel families that are activated by mechanical signals to direct intracellular events. One of such channel is formed by transient receptor potential cation channel subfamily V member, TRPV4 that is known to act as a mechanosensor in wide variaty of tissues and control ion-influx in a spatio-temporal way. Here we report that TRPV4 is prominently expressed in the stem/progenitor cell populations of the mammary epithelium and seems important for the lineage-specific differentiation, consequently affecting mechanical features of the mature mammary epithelium. This was evident by the lack of several markers for mature myoepithelial and luminal epithelial cells in TRPV4-depleted cell lines. Interestingly, TRPV4 expression is controlled in a tension-dependent manner and it also impacts differentation process dependently on the stiffness of the microenvironment. Furthermore, such cells in a 3D compartment were disabled to maintain normal mammosphere structures and displayed abnormal lumen formation, size of the structures and disrupted cellular junctions. Mechanosensitive TRPV4 channel therefore act as critical player in the homeostasis of normal mammary epithelium through sensing the physical environment and guiding accordingly differentiation and structural organization of the bilayered mammary epithelium.

A Conductive Microcavity Created by Assembly of Carbon Nanotube Buckypapers for Developing Electrochemically Wired Enzyme Cascades.

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H2O2 to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H2O2. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1-4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5-2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days.