Activity-based profiling of proteases.

Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2.

Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.

Refining hemodynamic correction in in vivo wide-field fluorescent imaging through linear regression analysis.

Accurate interpretation of in vivo wide-field fluorescent imaging (WFFI) data requires precise separation of raw fluorescence signals into neural and hemodynamic components. The classical Beer-Lambert law-based approach, which uses concurrent 530-nm illumination to estimate relative changes in cerebral blood volume (CBV), fails to account for the scattering and reflection of 530-nm photons from non-neuronal components leading to biased estimates of CBV changes and subsequent misrepresentation of neural activity. This study introduces a novel linear regression approach designed to overcome this limitation. This correction provides a more reliable representation of CBV changes and neural activity in fluorescence data. Our method is validated across multiple datasets, demonstrating its superiority over the classical approach.

Evaluation of a targeted anti-αvß3 integrin near-infrared fluorescent dye for fluorescence-guided resection of naturally occurring soft tissue sarcomas in dogs.

PURPOSE: Complete resection is a key prognostic factor for survival in patients with soft tissue sarcoma (STS), in humas and companion animals alike. Fluorescence-guided surgery could improve resection accuracy. As dogs are frequently affected by STS, they serve as a model to test an anti-αvß3 integrin targeting near-infrared fluorescent (NIRF) dye (AngiostampTM800) for fluorescence-guided surgery in STS to evaluate its safety and feasibility in dogs, and if it translates into a clinically relevant benefit compared to the standard of care with regards to completeness of surgery and local recurrence. Furthermore, we aimed to correlate target expression and NIRF-signal intensity. METHODS: Twenty dogs with STS were randomly allocated to either receive Angiostamp™ (NIRF group) or physiologic saline (control group) preoperatively. The researchers were blinded for treatment, and resections were adapted based on the NIRF-signal, if needed. Margin status was histologically determined at the 1 and 3 cm margin. The tumor-to-background ratio was measured in native tissue biopsies and formalin-fixed tissue. The fluorescent area was compared to the corresponding tumor areas as confirmed by histology using the Dice coefficient. Target expression was quantified by immunohistochemistry and correlated to NIRF-signal ratios. RESULTS: A fluorescent signal was detected in all 10 tumors of the NIRF group, with a tumor-to-background ratio of 7.4 ± 5.8 in native biopsies and 13.5 ± 10.9 in formalin-fixed tissue. In the NIRF group, resection margins were adapted in 5/10 cases, leading to complete resection and preventing R1 in four of these cases. In the NIRF and control group 9/10 and 8/10 resections were R0, with one local recurrence in each group and one sarcoma-related death in the NIRF group. The NIRF-signal correlated with the histologically confirmed tumor area (Dice coefficient 0.75 ± 0.17). Target expression was higher in tumor compared to peritumoral tissue (p < 0.0003) and showed a moderate correlation with the NIRF-signal (r = 0.6516, p < 0.0001). CONCLUSION: Fluorescence-guided surgery using Angiostamp™ can pinpoint residual disease in the tumor bed and contributes to an improved resection accuracy in canine STS.

An ESIPT-Based Fluorescent Probe for Wash-Free Cell Membrane Imaging.

The cell membrane tracking is important for studying the membrane function and diagnosing membrane-related diseases, so the development of fluorescent molecule specifically lighting up cell membrane is highly desirable. In this work, we designed and readily prepared a fluorescent dye (BOHI) that can target cell membrane. The pH-dependent emission spectra reveal that BOHI shows fluorescence based on the excited-state intramolecular proton transfer (ESIPT) under acidic and neutral conditions, while the blue-shifted emission from the phenolate anion of BOHI was observed when pH ≥ 9. Among various solvents, the weakest fluorescence emission was observed in H2O, which is favorable to wash-free imaging. The fluorescence intensity of BOHI is greatly affected by surfactants. The anionic surfactant can induce intense green fluorescence, while very weak fluorescence was observed in the solution of cationic surfactant. Hela cells images show that BOHI can specifically targets and lights up cell membrane.

Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging.

This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed in vitro, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for in vitro and in vivo imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or in vivo biological activities of the POI.

Design of a dual Ir-Eu tag for fluorescent visualization and ICP-MS quantification of SIRPα and its host cells.

With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153Eu/193Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 (153Eu) and 1.1 (193Ir) pM SIRPα and 220 (153Eu) and 830 (193Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable.

Chemical sensors detect and resolve proteome aggregation in peripheral neuropathy cell model induced by chemotherapeutic agents.

As a consequence of somatosensory nervous system injury or disease, neuropathic pain is commonly associated with chemotherapies, known as chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation in neuronal cells remain elusive due to limited detection tools. Herein, we present series sensors for fluorescence imaging (AggStain) and proteomics analysis (AggLink) to visualize and capture aggregated proteome in CIPN neuronal cell model. The environment-sensitive AggStain imaging sensor selectively binds and detects protein aggregation with 12.3 fold fluorescence enhancement. Further, the covalent AggLink proteomic sensor captures cellular aggregated proteins and profiles their composition via LC-MS/MS analysis. This integrative sensor platform reveals the presence of proteome aggregation in CIPN cell model and highlights its potential for broader applications in assessing proteome stability under various cellular stress conditions.

Monoamine oxidase B activatable red fluorescence probe for bioimaging in cells and zebrafish.

A real-time and specific for the detection of Monoamine Oxidase B (MAO-B) to investigate the MAO-B-relevant disease development and treatment process is urgently desirable. Here, we utilized MAO-B to catalyze the conversion of propylamino groups to aldehyde groups, which was then quickly followed by a ß-elimination process to produce fluorescent probes (FNJP) that may be used to detect MAO-B in vitro and in vivo. The FNJP probe possesses unique properties, including favorable reactivity (Km = 10.8 µM), high cell permeability, and NIR characteristics (λem = 610 nm). Moreover, the FNJP probe showed high selectivity for MAO-B and was able to detect endogenous MAO-B levels from a mixed population of NIH-3 T3 and HepG2 cells. MAO-B expression was found to be increased in cells under lipopolysaccharide-stimulated cellular oxidative stress in neuronal-like SH-SY5Y cells. In addition, the visualization of FNJP for MAO-B activity in zebrafish can be an effective tool for exploring the biofunctions of MAO-B. Considering these excellent properties, the FNJP probe may be a powerful tool for detecting MAO-B levels in living organisms and can be used for accurate clinical diagnoses of related diseases.

Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.

To date, 14C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14C/12C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Synthesis and Characterization of Sulfonamide-Containing Naphthalimides as Fluorescent Probes.

A tumor-targeting fluorescent probe has attracted increasing interest in fluorescent imaging for the noninvasive detection of cancers in recent years. Sulfonamide-containing naphthalimide derivatives (SN-2NI, SD-NI) were synthesized by the incorporation of N-butyl-4-ethyldiamino-1,8-naphthalene imide (NI) into sulfonamide (SN) and sulfadiazine (SD) as the tumor-targeting groups, respectively. These derivatives were further characterized by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (1H NMR), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV), and a fluorescence assay. In vitro properties, including cell cytotoxicity and the cell uptake of tumor cells, were also evaluated. Sulfonamide-containing naphthalimide derivatives possessed low cell cytotoxicity to B16F10 melanoma cells. Moreover, SN-2NI and SD-NI can be taken up highly by B16F10 cells and then achieve good green fluorescent images in B16F10 cells. Therefore, sulfonamide-containing naphthalimide derivatives can be considered to be the potential probes used to target fluorescent imaging in tumors.

A Red-Emission Fluorescent Probe with Large Stokes Shift for Detection of Viscosity in Living Cells and Tumor-Bearing Mice.

Abnormal viscosity is closely related to the occurrence of many diseases, such as cancer. Therefore, real-time detection of changes in viscosity in living cells is of great importance. Fluorescent molecular rotors play a critical role in detecting changes in cellular viscosity. Developing red emission viscosity probes with large Stokes shifts and high sensitivity and specificity remains an urgent and important topic. Herein, a novel viscosity-sensitive fluorescent probe (TCF-VIS1) with a large stokes shift and red emission was prepared based on the 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) skeleton. Due to intramolecular rotation, the probe itself does not fluorescence at low viscosity. With the increase in viscosity, the rotation of TCF-VIS1 is limited, and its fluorescence is obviously enhanced. The probe has the advantages of simple preparation, large Stokes shift, good sensitivity and selectivity, and low cytotoxicity, which make it successfully used for viscosity detection in living cells. Moreover, TCF-VIS1 showed its potential for cancer diagnosis at the cell level and in tumor-bearing mice by detecting viscosity. Therefore, the probe is expected to enrich strategies for the detection of viscosity in biological systems and offer a potential tool for cancer diagnosis.

Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing.

Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.

DOTA-Based Plectin-1 Targeted Contrast Agent Enables Detection of Pancreatic Cancer in Human Tissue.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC's first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria.

Strength in Numbers: A Giant NIR-II AIEgen with One-for-All Phototheranostic Features for Exceptional Orthotopic Bladder Cancer Treatment.

One-for-all phototheranostics that allows the simultaneous implementations of multiple optical imaging and therapeutic modalities by utilizing a single component, is growing into a sparkling frontier in cancer treatment. Of particular interest is phototheranostic agent with emission in the second near-infrared (NIR-II) window. Nevertheless, the practical uses of those conventional NIR-II agents are severely impeded by their unsatisfactory features including insufficient stability, low synthetic yield, to be extended absorption/ emission wavelengths, and inefficient phototheranostic outcomes. Developing exceptional phototheranostic agents is thus highly desirable yet remains formidably challenging. Herein, we synthesized two novel N-heteroacenes-based NIR-II luminogens, namely 2TT-PPT and 4TT-PBPT, by respectively employing pyrene-fused phenaziothiadiazoles and pyrene-fused bisphenaziothiadiazoles as acceptor skeletons. There is strength in numbers by increasing the fusing rings in N-heteroacenes moieties and numbers of appended donors. Compared to less ring-fused 2TT-PPT, the giant molecule 4TT-PBPT shows improved photophysical characteristics, such as enhanced light absorbance, red-shifted wavelengths, higher brightness, favorable reactive oxygen species (ROS) generation, and elevated photothermal conversion efficiency, which render 4TT-PBPT nanoparticles excellent fluorescence-photoacoustic-photothermal trimodal imaging guided photodynamic-photothermal synergistic therapy for orthotopic bladder cancer.

[Intraoperative near-infrared fluorescence imaging of peripheral lung tumors]. / Intraoperatsionnaya flyuorestsentnaya vizualizatsiya perifericheskikh opukholei legkikh v blizhnem infrakrasnom diapazone.

The main objective of surgical intervention in lung cancer is the radical removal of the entire tumor with the maximum possible preservation of healthy tissue. Although the area of the tumor lesion is known by the results of preoperative studies, it can be difficult to use this information to establish the exact boundaries of resection during surgery, especially with small sizes of the lump and when using minimally invasive approaches. There are several techniques to solve this problem. One of the latter is intraoperative fluorescence imaging in the infrared range, which makes it possible to detect a tumor not only with greater contrast than it can be done in white light, but also with its deep location. This review is devoted to the discussion of various aspects of this approach related to molecular imaging methods. The current situation based on the use of green indocyanine green, available for clinical use as a fluorescent agent is considered, the issues of using new targeted drugs are examined, as well as the possibility of increasing the depth of probing and combining with related treatment methods, which should contribute to a more radical operation and reduce the likelihood of local relapses.

[Prospects of qualitative and quantitative assessment of bowel perfusion by fluorescent angiography with indocyanine green in colorectal surgery. First experience]. / Perspektivy kachestvennoi i kolichestvennoi otsenki perfuzii kishki metodom flyuorestsentnoi angiografii s indotsianinom zelenym v kolorektal'noi khirurgii. Pervyi opyt.

OBJECTIVE: To assess the possibilities of fluorescent detection system in qualitative and quantitative assessment of bowel perfusion in colorectal resections. MATERIAL AND METHODS: From May to August 2023, a single-center pilot cross-sectional unblinded study with inclusion of 18 patients with colon cancer (of left-sided - 12, of right-sided - 6, mean age - 72.9 years, m/w - 61/39%) was conducted. All patients underwent laparoscopic colorectal resections with extracorporeal stage of bowel transection. The evaluation of the bowel's ICG perfusion was conducted to assist in decision making about the level of its resection. Qualitative (visual) assessment was carried out in all 18 patients, in one patient twice, quantitative assessment of perfusion was conducted in 8 patients (left-sided resections - 6, right-sided hemicolectomy - 2). The qualitative evaluation was performed in real time on the analysis of the color gradient. The time parameters and fluorescence intensity at different level proximally and distally from the resection line were quantitatively estimated: Tstart - time of occurrence of minimal fluorescence in the areas of interest after the ICG injection (sec); Tmax - time to achieve maximum fluorescence intensity after the ICG injection (sec); Tmax-start - time interval between Tstart and Tmax, Imax - level of maximum fluorescence intensity (I). RESULTS: Visual qualitative analysis of fluorescence revealed unsatisfactory perfusion characteristics (black, dark-gray color) in the area planned by the surgeon to anastomose the bowel in 3 of 18 patients (16.6%). When analyzing the quantitative data of this group of patients, there was a 2-6-fold decrease in Imax level, and one patient had an increase in Tmax-start at the level of intended resection compared to the bowel's sections in the favorable zone. In all cases, the final bowel transection was conducted in the area of good perfusion. There was no clinical evidence of anastomotic dehiscence in the study group. CONCLUSION: Intraoperative evaluation of bowel perfusion is an important component of safe anastomosis formation in colorectal surgery. The use of ICG-FA allows to conduct qualitative and quantitative assessment of tissue perfusion of the bowel in order to assist in making intraoperative decisions. Quantitative evaluation of fluorescence provides more objective information about perfusion parameters. Imax and Tmax-start are the most promising quantitative indicators of local bowel's perfusion. Nevertheless, the precise interpretation of the quantitative indicators of ICG perfusion needs to be clarified.

[Videofluorescence navigation during parenchymal-sparing liver resections using a domestic fluorescence imaging system]. / Videofluorestsentnaya navigatsiya pri parenkhimosokhrannykh rezektsiyakh pecheni s ispol'zovaniem otechestvennoi sistemy fluorestsentnoi vizualizatsii.

Parenchyma- sparing liver resections are aimed at maximizing the possible preservation of parenchyma not affected by the tumor - a current trend in hepatopancreatobiliary surgery. On the other hand, a prerequisite for operations is to ensure their radicality. To effectively solve this problem, all diagnostic imaging methods available in the arsenal are used, which make it possible to comprehensively solve the issues of perioperative planning of the volume and technical features of the planned operation. Diagnostic imaging methods that allow intraoperative navigation through intraoperative, instrumentally based determination of the tumor border and resection plane have additional value. One of the methods of such mapping is ICG video fluorescence intraoperative navigation. An analysis of the clinical use of the domestic video fluorescent navigation system "MARS" for parenchymal-sparing resections of focal liver lesions is presented. An assessment was made of the dynamics of the distribution of the contrast agent during ICG videofluorescent mapping during parenchymal-sparing resection interventions on the liver, with the analysis of materials from histological examination of tissues taking into account three-zonal videofluorescent marking of the resection edge, performed using the domestic videofluorescence imaging system «MARS¼.

Development of an Endoplasmic Reticulum-targeting Fluorescent Probe for the Imaging of Superoxide Anion in Living Cells.

Superoxide anion (O2•-) is an important reactive oxygen species (ROS), and plays critical roles in biological systems. ER stress has close relation with many metabolic diseases, and could lead to the abnormal production of ROS including O2•-. Herein, we present an ER-targeting probe (ER-Tf) for the detection of O2•- in living cells. The probe ER-Tf used triflate as the response site for O2•-, and employed p-methylbenzenesulfonamide as ER-targeting moiety. In response to O2•-, the triflate of the probe ER-Tf converted to hydroxyl group, providing strong blue emission under the excitation of ultraviolet light. The probe ER-Tf exhibited high sensitivity and selectivity to O2•-. Bioimaging experiments showed that the probe ER-Tf can be applied to detect O2•- at ER, and also demonstrated that rotenone could increase the generation of O2•- in living cells, while the O2•- level at ER showed no remarkable change during ferroptosis.

Near-infrared fluorescence imaging with indocyanine green to assess the blood supply of the reconstructed gastric conduit to reduce anastomotic leakage after esophagectomy: a literature review.

The blood supply of the right gastroepiploic artery after esophagectomy with gastric tube reconstruction is essential for avoiding anastomotic leakage. Near-infrared fluorescence (NIRF) imaging with indocyanine green is widely used to assess the blood supply because it can visualize it in real-time during navigation surgery. However, there is no established protocol for this modality. One reason for this lack of protocol is that NIRF provides subjective information. This study aimed to evaluate NIRF quantification. We conducted a literature review of risk factors for anastomotic leakage after esophagectomy, NIRF procedures, NIRF quantification, and new methods to compensate for NIRF limitations. Major methods for the quantification of NIRF include measuring the blood flow speed, visualization time, and fluorescence intensity. The cutoff value for the blood flow speed is 2.07 cm/s, and that for the visualization time is 30-90 s. Although the time-intensity curve provided patterns of change in the blood flow, it did not show an association with anastomotic leakage. However, to compensate for the limitations of NIRF, new devices have been reported that can assess tissue oxygenation perfusion, organ hemoglobin concentration, and microcirculation.