Active Probe Research Articles

Functional 20S Proteasomes in Retroviruses: Evidence in Favor.

Proteasomes are barrel-like cellular protein complexes responsible for the degradation of most intracellular proteins. Earlier, it has been shown that during assembly, hundreds of different cellular proteins are incorporated into retro-and herpes viruses. Among detected cellular proteins, there were different proteasome subunits (PS). Previous reports postulated the incorporation of 20S proteasome subunits and subunits of proteasome regulator complexes inside retroviruses. Here, we demonstrated the association of functional 20S proteasome with gammaretroviruses, betaretroviruses, and lentiviruses. Cleaved proteasome subunits β1, β2 and β5 were detected in tested viruses. Using fluorescent peptides and a cell-permeable proteasome activity probe, proteasome activity was detected in endogenous and exogenous retroviruses, including recombinant HIV-1. Taken together, our data favors the insertion of functional proteasomes into the retroviruses during assembly. The possible role of proteasomes in retroviruses is discussed.

Identification of Aberrant Expression of Gemcitabine-Targeting Proteins in Drug-Resistant Cells Using an Activity-Based Gemcitabine Probe.

Gemcitabine-based monotherapy or combination therapy has become the standard treatment for locally advanced and metastatic pancreatic cancer. However, the emergence of resistance within weeks of treatment severely compromises therapeutic efficacy. The intricate biological process of gemcitabine resistance in pancreatic cancer presents a complex challenge, as the underlying mechanisms remain unclear. Identifying the target protein of gemcitabine is crucial for studying its drug-resistance mechanism. An activity-based probe is a powerful tool for studying drug target proteins, but the current lack of activity-based gemcitabine probes with robust biological activity hinders research on gemcitabine. In this study, we developed three active probes based on gemcitabine, among which Gem-3 demonstrated excellent stability and labeling efficacy. We utilized Gem-3 in conjunction with chemical proteomics to identify intracellular target proteins. We identified 79 proteins that interact with gemcitabine, most of which were previously unknown and represented various functional classes. Additionally, we validated the increased expression of IFIT3 and MARCKS in drug-resistant cells, along with the activation of the NF-κB signaling pathway. These findings substantially contribute to our comprehension of gemcitabine's target proteins and further our understanding of the mechanisms driving gemcitabine resistance in pancreatic cancer cells.

Cathepsin B- and L-like Protease Activities Are Induced During Developmental Barley Leaf Senescence.

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. 'GemCraft') leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC50 values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC50 in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves.

Single-cell chemoproteomics identifies metastatic activity signatures in breast cancer.

Protein activity state, rather than protein or mRNA abundance, is a biologically regulated and relevant input to many processes in signaling, differentiation, development, and diseases such as cancer. While there are numerous methods to detect and quantify mRNA and protein abundance in biological samples, there are no general approaches to detect and quantify endogenous protein activity with single-cell resolution. Here, we report the development of a chemoproteomic platform, single-cell activity-dependent proximity ligation, which uses automated, microfluidics-based single-cell capture and nanoliter volume manipulations to convert the interactions of family-wide chemical activity probes with native protein targets into multiplexed, amplifiable oligonucleotide barcodes. We demonstrate accurate, reproducible, and multiplexed quantitation of a six-enzyme (Ag-6) panel with known ties to cancer cell aggressiveness directly in single cells. We further identified increased Ag-6 enzyme activity across breast cancer cell lines of increasing metastatic potential, as well as in primary patient-derived tumor cells and organoids from patients with breast cancer.

A Cascade Activation Probe with Double-Enhanced Near-Infrared Imaging for Monitoring Peroxynitrite Fluctuations in Vivo.

Monitoring peroxynitrite (ONOO-) fluctuations is particularly important for assessing pathological progression and oxidative damage due to their crucial role in maintaining the redox balance of organisms. However, due to the lack of efficient tools for differentially monitoring ONOO- fluctuations at different concentration ranges in vivo, the precise detection of endogenous ONOO- fluctuations under pathological conditions in living systems remains challenging. Herein, we rationally designed a double-enhanced emission cascade activatable near-infrared (NIR) fluorescent probe (B-TCF) for the measurement of ONOO-, which consists of a borate ester response group and a malononitrile hemicyanine fluorophore. Especially, after sequential oxidative hydrolysis of the borate ester group and xanthene skeleton, B-TCF exhibited a sequentially double-enhanced NIR emission response at 776 and 625 nm for different ONOO- concentration ranges. Moreover, B-TCF revealed excellent and promising performance for ONOO- in terms of high selectivity, sensitivity, and reaction rate (k = 28.2 M-1 s-1). Motivated by the two-step emission signal enhancement and large wavelength shift in the NIR region, B-TCF enabled discriminative imaging of ONOO- with the low and high concentrations in living cells. Importantly, B-TCF was successfully applied for assessing the pathological progression of isoniazid and acetaminophen-induced liver damage in vivo by detecting the endogenous different ONOO- levels. Overall, this study not only demonstrates the first double-enhanced emission cascade activatable NIR fluorescent probe for measuring the dynamic variation of ONOO- in related diseases but also shows great potential as an effective molecular tool for evaluating the various stages of drug-induced liver damage.

High Impedance Active Probe for High Voltages

The probe was designed for the measurement of DC voltages of up to 30 kV from high impedance sources. It is based on a resistive divider with a total resistance of 200 GΩ and a step-down factor of 10’000. In order to allow the measurement of the stepped down voltage with a conventional multimeter without loading, the signal was buffered with an operational amplifier. The device was calibrated against a commercial probe using a low impedance high voltage source. A linear relationship was obtained for a high impedance resistive ladder for voltages between 3 and 30 kV, with a coefficient of determination (R2) of 0.9999. The low-cost device (ca. US $200) fills an application niche not addressed by commercial products.

Exploration of glyoxals’ level changes in endoplasmic reticulum during ferroptosis by a photocaged fluorescent probe

Both ferroptosis and elevated glyoxals (e.g. methylglyoxal and glyoxal), are associated with endoplasmic reticulum stress, but their correlations, in particular, how level of glyoxals changes in endoplasmic reticulum during ferroptosis remains unclear. In this work, a new photoactivatable fluorescent probe Photo-ER-GOS was developed to study level changes of glyoxals in endoplasmic reticulum during ferroptosis via in situ photodecaging and fluorescence detection, avoiding potential interference from glyoxals in cytosol. The probe contains a photolabile nitrobenzyloxymethyl group functioned both as the reactivity blocking group and the fluorescence quenching group, which linked to the guanidino group of NAP-DCP-3, the active endoplasmic reticulum-targeting fluorescent probe for glyoxals previously developed in our lab. It was found that the level of glyoxals in endoplasmic reticulum increases significantly during erastin-induced ferroptosis in RAW 264.7 cells via comparison of relative fluorescence intensity increases. The endoplasmic reticulum glyoxal level increase was also confirmed by the Ultra Performance Liquid Chromatography-Mass Spectrum studies. In contrast, no significant change of level of glyoxals in endoplasmic reticulum was found in staurosporine-induced apoptosis, suggesting that upregulated glyoxals in endoplasmic reticulum may be a previously overlooked characteristic of ferroptosis. Erastin-induced level increase of glyoxals was also found in zebrafish. The caged probe Photo-ER-GOS thus provides a useful chemical tool to study GOS levels changes in endoplasmic reticulum.

Synthesis and Application of a Versatile Immunoproteasome Activity Probe.

The immunoproteasome (iCP) has gained significant interest in recent years as it has been discovered to be significantly expressed under inflammatory conditions, as well as playing significant roles in several diseases, such as autoimmune disorders, viral infection, and cancer. Selective inhibitors have been generated as a method to overcome the off-target effects of current proteasome inhibitor therapeutics. However, selective probes that allow for monitoring this protein complex remain limited, hindering our understanding of the iCP. Current probes are non-selective, not commercially available, or require difficult synthesis. Here, we describe the modular synthesis and application of an iCP-selective probe. The modular nature of the synthetic strategy can enable the incorporation of different fluorophores and covalent warheads, demonstrating the versatility of this probe.

Multifunctional BiOCl: Sm3+ nanophosphors: A luminescent platform for solid-state lighting applications, optical thermometry, photocatalytic and forensic applications

Rare earth-activated phosphor materials have been widely used as active probes in optical materials for multidimensional applications. Here, we report a series of ultrahigh purity orange-red emitting BiOCl: xSm3+ (x= 0.5- 2.5 mol %) nanophosphors synthesized via conventional solid-state method at relatively low temperatures for various photonic and forensic applications. XRD and Rietveld refinement results confirmed the tetragonal crystal structure of the synthesized phosphor materials with space group P4/nmm. Fourier transform infrared (FT-IR) and Raman spectroscopy were used to perform the functional group analysis. The UV-Vis-NIR spectra were analyzed to determine the Judd-Ofelt (JO) intensity parameters (Ω2, Ω4, Ω6) and to identify the bonding structure and spontaneous emission properties of the samples. The prepared phosphors exhibit intense orange-red emission under 408 nm excitation and beyond 1 mol% doping of Sm3+ ions, the emission intensity decreases due to concentration quenching mechanism via, dipole-dipole interaction between the optically active Sm3+ ions in the phosphor sample. The synthesized BiOCl: Sm3+ nanophosphors possess low correlated color temperature (CCT), admirable high color purity (99.4%) and outstanding internal quantum efficiency (IQE) of 95.27%. The optical thermometry behavior of the optimized sample has been investigated in detail. Moreover, the optimized nanophosphor stains on porous and non-porous surfaces show clear ridge patterns with high sensitivity, efficiency, and minimal background hindrance for latent fingerprints and lip print detection in forensic applications and are used as security ink in anti-counterfeiting applications. Overall, the results revealed the potential prospects of BiOCl: Sm3+ nanophosphors in the field of display applications, optical thermometry, and forensic sciences.

TrojanProbe: Fingerprinting Trojan tunnel implementations by actively probing crafted HTTP requests

Trojan is a well-known hidden tunnel protocol widely used to bypass Internet censorship and thus presents a big challenge to transparent network management and forensics. As claimed by the protocol designer, Trojan maintains its anti-identifiability by proxying real HTTPS/TLS traffic to react to unauthenticated requests, eliminating any subtle differences between the Trojan traffic and the legitimate HTTPS. Despite such a protocol seeming unidentifiable by design, the diverse Trojan implementations adopting very different programming languages will likely have varied coding logic and networking API calls, opening a new door to be identified and fingerprinted from the implementation level. In this paper, we propose TrojanProbe, a new class of active probing methods that can be used to fingerprint Trojan implementations by triggering their identifiable responses. Our basic idea is to audit the source code of the Trojan programs and discover the subtle logic discrepancy compared with the legitimate HTTPS counterparts, to craft specific HTTP requests as probes to trigger these differences for fingerprinting. By this idea, we choose the five most popular open-source Trojan programs off-the-shelf as our targets to audit, covering the majority of Trojan market share and the mainstream programming languages from traditional C++ to the cutting-edge Go and Rust, and design a suite of novel HTTP probes to differentiate them from their web server masquerades. Our probes exploit either the different responding/buffering logic to the malformed HTTP requests and the different HTTP versions, or the varied timeouts set in the different networking APIs by default. To this end, we have conducted extensive experiments to evaluate the TrojanProbe against a comprehensive set of configuration and networking conditions. The experimental results show that our TrojanProbe can effectively fingerprint our selected Trojan targets in most conditions, but leave a single Rust implementation with a minority market occupied that can only be identified in some constraint cases. Despite such an exception, our research sheds light on a new kind of possibility to fingerprint Trojans at their implementation level, even if such a hidden tunnel is widely known as unidentifiable at the protocol level.

ID: 340650 Active electrical probing of brain networks to characterize connectivity for novel deep brain stimulation

ID: 340650 Active electrical probing of brain networks to characterize connectivity for novel deep brain stimulation

Development of an Activity-Based Ratiometric Electrochemical Switch for Direct, Real-Time Sensing of Pantetheinase in Live Cells, Blood, and Urine Samples.

Pantetheinase is a key biomarker for the diagnosis of acute kidney injury and the monitoring of malaria progression. Currently, existing methods for sensing pantetheinase, also known as Vanin-1, show considerable potential but come with certain limitations, including their inability to directly sense analytes in turbid biofluid samples without tedious sample pretreatment. Here, we describe the first activity-based electrochemical probe, termed VaninLP, for convenient and specific direct targeting of pantetheinase activity in turbid liquid biopsy samples. The probe was designed such that cleavage of the pantetheinase amide linkage, triggered by a self-immolative reaction, simultaneously ejects an amino ferrocene reporter. Among the distinctive properties of the VaninLP probe for sensing pantetheinase are its high selectivity, sensitivity, and enzyme affinity, a wide linear concentration range (8-300 ng/mL), and low limit of detection (2.47 ng/mL). The designed probe precisely targeted pantetheinase and was free of interference by other electroactive biological species. We further successfully applied the VaninLP probe to monitor and quantify the activity of pantetheinase on the surfaces of HepG2 tumor cells, blood, and urine samples. Collectively, our findings indicate that VaninLP holds significant promise as a point-of-care tool for diagnosing early-stage kidney injury, as well as monitoring the progression of malaria.

A General Method for Calibration of Active Scanning Thermal Probes

Scanning thermal microscopy (SThM) is a scanning probe technique aimed at quantitative characterization of local thermal properties at the length scale down to tens of nanometers. With many probe designs and approaches to interpretation of probe responses, there is a need for a universal framework, which would allow probe calibration and comparison of probe performance. Herein, a calibration framework based on an abstracted, formal, probe model for active SThM probes is developed. The calibration can be accomplished through measurements with two or three calibration samples. Requirements for calibration samples are described with examples of structures of suitable samples identified in published literature. A link to a published experimental work indirectly verifying the proposed procedure is provided. The calibration does not require knowledge of internal probe properties and yields a small and universal set of parameters that can be used to quantify thermal resistance presented to the probe by samples as well as to characterize active‐mode SThM probes of any type and at any measurement frequency. How the probe calibration parameters can be used to guide probe design is illustrated. When the calibration approach can be used directly to measure the thermal conductivity of unknown samples is also analyzed.

Novel sulfonylurea fluorescence probes for antiproliferative activity, detection and imaging of fluoride ions in living cells

In this study, four novel triphenylpyrazoline 4-toluenesulfonylureas (PYSUs) were synthesized to develop potential anticancer drugs and fluorescent probes for the detection of F− ions. The compounds were synthesized via a two-step microwave-assisted method. The antiproliferative activity of the PYSUs was tested against two cancer cell lines: HepG2 and MDA-MB-231. Among the compounds, 3a exhibited strong activity against both cell lines, while 3b showed strong activity against MDA-MB-231 cells but weaker inhibition of HepG2 cells. Furthermore, compound 3b was identified as the best fluorescent probe, with an increase in emission intensity in the presence of F− ions from tetrabutylammonium fluoride (TBAF). In DMSO, the emission wavelength was found to be 518 nm (green) with an excitation wavelength of 469 nm. The limit of detection (LOD) was calculated to be 0.269 mM in the linear range of 5–10 mM. The interaction mechanism between F− ions and compound 3b was further explored via DFT/TDDFT calculations. Additionally, fluorescent imaging of TBAF-incubated MDA-MB-231 and HepG2 cells confirmed the potential of PYSUs as fluorescent sensors for F− ions in living cancer cells.

Optical photothermal infrared imaging using metabolic probes in biological systems.

Infrared spectroscopy is a powerful tool for identifying biomolecules. In biological systems, infrared spectra provide information on structure, reaction mechanisms, and conformational change of biomolecules. However, the promise of applying infrared imaging to biological systems has been hampered by low spatial resolution and the overwhelming water background arising from the aqueous nature of in cell and in vivo work. Recently, optical photothermal infrared microscopy (OPTIR) has overcome these barriers and achieved both spatially and spectrally resolved images of live cells and organisms. Here, we determine the most effective modes of collection for work in biological samples. We examine three cell lines (Huh-7, differentiated 3T3-L1, and U2OS) and three organisms ( E. coli , tardigrades, and zebrafish). Our results suggest that the information provided by multifrequency imaging is comparable to hyperspectral imaging while reducing imaging times twenty-fold. We also explore the utility of IR active probes, including global and site-specific probes, for tracking metabolic pathways and protein localization, structure, and local environment. Our findings illustrate the versatility of OPTIR, and together, provide a direction for future dynamic imaging of living cells and organisms.

Effect of protein binding on the pharmacokinetics of the six substrates in the Basel phenotyping cocktail in healthy subjects and patients with liver cirrhosis

Phenotyping serves to estimate enzyme activities in healthy persons and patients in vivo. Low doses of enzyme-specific substrates are administered, and activities estimated using metabolic ratios (MR, calculated as AUCmetabolite/AUCparent). We administered the Basel phenotyping cocktail containing caffeine (CYP1A2 substrate), efavirenz (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6) and midazolam (CYP3A) to 36 patients with liver cirrhosis and 12 control subjects and determined free and total plasma concentrations over 24 h. Aims were to assess whether MRs reflect CYP activities in patients with liver cirrhosis and whether MRs calculated with free plasma concentrations (MRfree) provide better estimates than with total concentrations (MRtotal). The correlation of MRtotal with MRfree was excellent (R2 >0.910) for substrates with low (<30 %, caffeine and metoprolol) and intermediate protein binding (≥30 and <99 %, midazolam and omeprazole) but weak (R2 <0.30) for substrates with high protein binding (≥99 %, efavirenz and flurbiprofen). The correlations between MRtotal and MRfree with CYP activities were good (R2 >0.820) for CYP1A2, CYP2C19 and CYP2D6. CYP3A4 activity was reflected better by midazolam elimination than by midazolam MRtotal or MRfree. The correlation between MRtotal and MRfree with CYP activity was not significant or weak for CYP2B6 and CYP2C9. In conclusion, MRs of substrates with an extensive protein binding (>99 %) show high inter-patient variabilities and do not accurately reflect CYP activity in patients with liver cirrhosis. Protein binding of the probe drugs has a high impact on the precision of CYP activity estimates and probe drugs with low or intermediate protein binding should be preferred.

An integrated bipolar electrode with shared cathode for dual-mode detection and imaging of CEA

In this paper, we designed a novel shared cathode bipolar electrode chip based on Ohm 's law and successfully constructed a dual-mode dual-signal biosensor platform (DD-cBPE). The device integrates ELISA, ECL, and ECL imaging to achieve highly sensitive detection and visual imaging of carcinoembryonic antigen (CEA). The unique circuit structure of the device not only realizes the dual signal detection of the target, but also breaks the traditional signal amplification concept. The total resistance of the system is reduced by series-parallel connection of BPE, and the total current in the circuit is increased. In addition, Au@NiCo2O4@MnO2 nanozyme activity probe was introduced into the common cathode to enhance the conductivity of the material. At the same time, due to the excellent peroxidase (POD) activity of NiCo2O4@MnO2, the decomposition of H2O2 was accelerated, so that more electrons flowed to the BPE anode, and finally the dual amplification of the ECL signal was realized. The device affects the current in the circuit by regulating the concentration of the co-reactant TPrA, thereby affecting the resistance of the system. Finally, different luminescent reagents emit light at the same potential and the luminous efficiency is similar. In addition, the chip does not need external resistance regulation, which improves the sensitivity of the immunosensor and meets the needs of timely detection. It provides a new idea for the deviceization of bipolar electrodes and has broad application prospects in biosensors, clinical detection, and environmental monitoring.

A voltammetrically potentionmetric pH sensor based on multiply hydroxylated porphyrin

AbstractUsing voltammetry to readout potentiometrically the solution acidity requires easy pH tunability of the probe's redox properties. In this work, tri‐hydroxyphenyl porphyrin (POH3) was synthesized as the electrochemically active probe to develop a voltammetrically potentiometric sensor (VPS) with a pH‐sensitive response. By comparing POH3 with control di‐ and mono‐hydroxyphenyl porphyrins (POH2 and POH1), we found that protonation of the pyrrole macrocycle, electron communication of the para‐hydroxyl groups with the pyrrole nitrogens during structure tautomerization, and the presence of multiple ionizable hydroxyl groups are the critical factors to report reversibly the pH‐sensitive VPS response at the appropriate potential range. Common metal ions have negligible effects on the sensor performance. In comparison to the conventional potentiometric sensor (CPS), our VPS strategy benefits from the advantage that the electrode modification is not required. Thus, the complex phase boundary models necessary for CPS are unnecessarily involved in explaining the potential response.

Cross-Priming-Linked Hierarchical Isothermal Amplification Programming Progressive Activating Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a in miRNA Signaling.

Cascade isothermal nucleic acid amplification, which integrates several different amplification protocols to enhance the assay performance, is widely utilized in biosensing, particularly for detecting microRNAs (miRNAs), crucial biomarkers associated with tumor initiation and progression. However, striking a balance between a high amplification efficiency and simplicity in design remains a challenge. Therefore, methods achieving high amplification efficiency without significantly increasing complexity are highly favored. In this study, we propose a novel approach for miRNA detection, employing cross-priming-linked hierarchical isothermal amplification (CP-HIA) to progressively activate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. The CP-HIA method strategically combines nicking-rolling circle amplification (n-RCA) and palindrome-aided circular strand displacement amplification (p-CSDA) for miRNA detection. Remarkably, this method utilizes only two main probes. Its key innovation lies in the interactive cross-priming strategy, wherein the amplification product from n-RCA is recycled to further drive p-CSDA, and vice versa. This interactive process establishes a hierarchical amplification, significantly enriching the activation probes for progressive CRISPR/Cas12a activation and subsequent target signal amplification. Consequently, the method exhibits greatly enhanced analytical performance, including high sensitivity and specificity in detecting low concentrations of miRNA. As low as 1.06 fM miRNA can thus be quantitatively detected, and the linear response of the miRNA is from 10 fM to 10 nM. These features demonstrate its potential for early disease diagnosis and monitoring. We anticipate that the CP-HIA method will serve as a promising platform for developing advanced molecular diagnostic tools for biomedical research.

The critical dynamics of hippocampal seizures

Epilepsy is defined by the abrupt emergence of harmful seizures, but the nature of these regime shifts remains enigmatic. From the perspective of dynamical systems theory, such critical transitions occur upon inconspicuous perturbations in highly interconnected systems and can be modeled as mathematical bifurcations between alternative regimes. The predictability of critical transitions represents a major challenge, but the theory predicts the appearance of subtle dynamical signatures on the verge of instability. Whether such dynamical signatures can be measured before impending seizures remains uncertain. Here, we verified that predictions on bifurcations applied to the onset of hippocampal seizures, providing concordant results from in silico modeling, optogenetics experiments in male mice and intracranial EEG recordings in human patients with epilepsy. Leveraging pharmacological control over neural excitability, we showed that the boundary between physiological excitability and seizures can be inferred from dynamical signatures passively recorded or actively probed in hippocampal circuits. Of importance for the design of future neurotechnologies, active probing surpassed passive recording to decode underlying levels of neural excitability, notably when assessed from a network of propagating neural responses. Our findings provide a promising approach for predicting and preventing seizures, based on a sound understanding of their dynamics.