Observing bioorthogonal macrocyclizations in the nuclear envelope of live cells using on/on fluorescence lifetime microscopy.

The reactive partnership between azides and strained alkynes is at the forefront of bioorthogonal reactions, with their in situ cellular studies often achieved through the use of off to on fluorophores with fluorescence microscopy. In this work, the first demonstration of a bioorthogonal, macrocycle-forming reaction occurring within the nuclear envelope of live cells has been accomplished, utilising on/on fluorescence lifetime imaging microscopy for real-time continuous observation of the transformation. The fluorescent, macrocyclic BF2 azadipyrromethene was accessible through a double 1,3-dipolar cycloaddition within minutes, between a precursor bis-azido substituted fluorophore and Sondheimer diyne in water or organic solvents. Photophysical properties of both the starting bis-azide BF2 azadipyrromethene and the fluorescent macrocyclic products were obtained, with near identical emission wavelengths and intensities, but different lifetimes. In a novel approach, the progress of the live-cell bioorthogonal macrocyclization was successfully tracked through a fluorescence lifetime change of 0.6 ns from starting material to products, with reaction completion achieved within 45 min. The continuous monitoring and imaging of this bioorthogonal transformation in the nuclear membrane and invaginations, of two different cancer cell lines, has been demonstrated using a combination of fluorescence intensity and lifetime imaging with phasor plot analysis. As there is a discernible difference in fluorescence lifetimes between starting material and products, this approach removes the necessity for off-to-on fluorogenic probes when preparing for bioorthogonal cell-imaging and microscopy.

Conditions Leading to Ketene Formation in Vaping Devices and Implications for Public Health.

The outbreak of e-cigarette or vaping use-associated lung injury (EVALI) in the United States in 2019 led to a total of 2807 hospitalizations with 68 deaths. While the exact causes of this vaping-related lung illness are still being debated, laboratory analyses of products from victims of EVALI have shown that vitamin E acetate (VEA), an additive in some tetrahydrocannabinol (THC)-containing products, is strongly linked to the EVALI outbreak. Because of its similar appearance and viscosity to pure THC oil, VEA was used as a diluent agent in cannabis oils in illicit markets. A potential mechanism for EVALI may involve VEA's thermal decomposition product, ketene, a highly poisonous gas, being generated under vaping conditions. In this study, a novel approach was developed to evaluate ketene production from VEA vaping under measurable temperature conditions in real-world devices. Ketene in generated aerosols was captured by two different chemical agents and analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method takes advantage of the high sensitivity and specificity of tandem mass spectrometry and appears to be more suitable than GC-MS for the analysis of large batches of samples. Our results confirmed the formation of ketene when VEA was vaped. The production of ketene increased with repeat puffs and showed a correlation to temperatures (200 to 500 °C) measured within vaping devices. Device battery power strength, which affects the heating temperature, plays an important role in ketene formation. In addition to ketene, the organic oxidant duroquinone was also obtained as another thermal degradation product of VEA. Ketene was not detected when vitamin E was vaped under the same conditions, confirming the importance of the acetate group for its generation.

A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Forecasting vaping health risks through neural network model prediction of flavour pyrolysis reactions.

Vaping involves the heating of chemical solutions (e-liquids) to high temperatures prior to lung inhalation. A risk exists that these chemicals undergo thermal decomposition to new chemical entities, the composition and health implications of which are largely unknown. To address this concern, a graph-convolutional neural network (NN) model was used to predict pyrolysis reactivity of 180 e-liquid chemical flavours. The output of this supervised machine learning approach was a dataset of probability ranked pyrolysis transformations and their associated 7307 products. To refine this dataset, the molecular weight of each NN predicted product was automatically correlated with experimental mass spectrometry (MS) fragmentation data for each flavour chemical. This blending of deep learning methods with experimental MS data identified 1169 molecular weight matches that prioritized these compounds for further analysis. The average number of discrete matches per flavour between NN predictions and MS fragmentation was 6.4 with 92.8% of flavours having at least one match. Globally harmonized system classifications for NN/MS matches were extracted from PubChem, revealing that 127 acute toxic, 153 health hazard and 225 irritant classifications were predicted. This approach may reveal the longer-term health risks of vaping in advance of clinical diseases emerging in the general population.

A thiol-ene mediated approach for peptide bioconjugation using 'green' solvents under continuous flow.

Flow chemistry has emerged as an integral process within the chemical sector permitting energy efficient synthetic scale-up while improving safety and minimising solvent usage. Herein, we report the first applications of the photoactivated, radical-mediated thiol-ene reaction for peptide bioconjugation under continuous flow. Bioconjugation reactions employing deep eutectic solvents, bio-based solvents and fully aqueous systems are reported here for a range of biologically relevant peptide substrates. The use of a water soluble photoinitiator, Irgacure 2959, permitted synthesis of glycosylated peptides in fully aqueous conditions, obviating the need for addition of organic solvents and enhancing the green credentials of these rapid, photoactivated, bioconjugation reactions.

BF2-Azadipyrromethene Fluorophores for Intraoperative Vital Structure Identification.

A series of mono- and bis-polyethylene glycol (PEG)-substituted BF2-azadipyrromethene fluorophores have been synthesized with emissions in the near-infrared region (700-800 nm) for the purpose of fluorescence guided intraoperative imaging; chiefly ureter imaging. The Bis-PEGylation of fluorophores resulted in higher aqueous fluorescence quantum yields, with PEG chain lengths of 2.9 to 4.6 kDa being optimal. Fluorescence ureter identification was possible in a rodent model with the preference for renal excretion notable through comparative fluorescence intensities from the ureters, kidneys and liver. Ureteral identification was also successfully performed in a larger animal porcine model under abdominal surgical conditions. Three tested doses of 0.5, 0.25 and 0.1 mg/kg all successfully identified fluorescent ureters within 20 min of administration which was sustained up to 120 min. 3-D emission heat map imaging allowed the spatial and temporal changes in intensity due to the distinctive peristaltic waves of urine being transferred from the kidneys to the bladder to be identified. As the emission of these fluorophores could be spectrally distinguished from the clinically-used perfusion dye indocyanine green, it is envisaged that their combined use could be a step towards intraoperative colour coding of different tissues.

Refining Glioblastoma Surgery through the Use of Intra-Operative Fluorescence Imaging Agents.

Glioblastoma (GBM) is the most aggressive adult brain tumour with a dismal 2-year survival rate of 26-33%. Maximal safe resection plays a crucial role in improving patient progression-free survival (PFS). Neurosurgeons have the significant challenge of delineating normal tissue from brain tumour to achieve the optimal extent of resection (EOR), with 5-Aminolevulinic Acid (5-ALA) the only clinically approved intra-operative fluorophore for GBM. This review aims to highlight the requirement for improved intra-operative imaging techniques, focusing on fluorescence-guided imaging (FGS) and the use of novel dyes with the potential to overcome the limitations of current FGS. The review was performed based on articles found in PubMed an.d Google Scholar, as well as articles identified in searched bibliographies between 2001 and 2022. Key words for searches included 'Glioblastoma' + 'Fluorophore'+ 'Novel' + 'Fluorescence Guided Surgery'. Current literature has favoured the approach of using targeted fluorophores to achieve specific accumulation in the tumour microenvironment, with biological conjugates leading the way. These conjugates target specific parts overexpressed in the tumour. The positive results in breast, ovarian and colorectal tissue are promising and may, therefore, be applied to intracranial neoplasms. Therefore, this design has the potential to produce favourable results in GBM by reducing the residual tumour, which translates to decreased tumour recurrence, morbidity and ultimately, mortality in GBM patients. Several preclinical studies have shown positive results with targeted dyes in distinguishing GBM cells from normal brain parenchyma, and targeted dyes in the Near-Infrared (NIR) emission range offer promising results, which may be valuable future alternatives.

Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring.

The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.

Digital dynamic discrimination of primary colorectal cancer using systemic indocyanine green with near-infrared endoscopy.

As indocyanine green (ICG) with near-infrared (NIR) endoscopy enhances real-time intraoperative tissue microperfusion appreciation, it may also dynamically reveal neoplasia distinctively from normal tissue especially with video software fluorescence analysis. Colorectal tumours of patients were imaged mucosally following ICG administration (0.25 mg/kg i.v.) using an endo-laparoscopic NIR system (PINPOINT Endoscopic Fluorescence System, Stryker) including immediate, continuous in situ visualization of rectal lesions transanally for up to 20 min. Spot and dynamic temporal fluorescence intensities (FI) were quantified using ImageJ (including videos at one frame/second, fps) and by a bespoke MATLAB® application that provided digitalized video tracking and signal logging at 30fps (Fluorescence Tracker App downloadable via MATLAB® file exchange). Statistical analysis of FI-time plots compared tumours (benign and malignant) against control during FI curve rise, peak and decline from apex. Early kinetic FI signal measurement delineated discriminative temporal signatures from tumours (n = 20, 9 cancers) offering rich data for analysis versus delayed spot measurement (n = 10 cancers). Malignant lesion dynamic curves peaked significantly later with a shallower gradient than normal tissue while benign lesions showed significantly greater and faster intensity drop from apex versus cancer. Automated tracker quantification efficiently expanded manual results and provided algorithmic KNN clustering. Photobleaching appeared clinically irrelevant. Analysis of a continuous stream of intraoperatively acquired early ICG fluorescence data can act as an in situ tumour-identifier with greater detail than later snapshot observation alone. Software quantification of such kinetic signatures may distinguish invasive from non-invasive neoplasia with potential for real-time in silico diagnosis.

Correlation of national and healthcare workers COVID-19 infection data; implications for large-scale viral testing programs.

Time analysis of the course of an infectious disease epidemic is a critical way to understand the dynamics of pathogen transmission and the effect of population scale interventions. Computational methods have been applied to the progression of the COVID-19 outbreak in five different countries (Ireland, Germany, UK, South Korea and Iceland) using their reported daily infection data. A Gaussian convolution smoothing function constructed a continuous epidemic line profile that was segmented into longitudinal time series of mathematically fitted individual logistic curves. The time series of fitted curves allowed comparison of disease progression with differences in decreasing daily infection numbers following the epidemic peak being of specific interest. A positive relationship between the rate of declining infections and countries with comprehensive COVID-19 testing regimes existed. Insight into different rates of decline infection numbers following the wave peak was also possible which could be a useful tool to guide the reopening of societies. In contrast, extended epidemic timeframes were recorded for those least prepared for large-scale testing and contact tracing. As many countries continue to struggle to implement population wide testing it is prudent to explore additional measures that could be employed. Comparative analysis of healthcare worker (HCW) infection data from Ireland shows it closely related to that of the entire population with respect to trends of daily infection numbers and growth rates over a 57-day period. With 31.6% of all test-confirmed infections in healthcare workers (all employees of healthcare facilities), they represent a concentrated 3% subset of the national population which if exhaustively tested (regardless of symptom status) could provide valuable information on disease progression in the entire population (or set). Mathematically, national population and HCWs can be viewed as a set and subset with significant influences on each other, with solidarity between both an essential ingredient for ending this crisis.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery. Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen. Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing. Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

Practical Perfusion Quantification in Multispectral Endoscopic Video: Using the Minutes after ICG Administration to Assess Tissue Pathology.

The wide availability of near infrared light sources in interventional medical imaging stacks enables non-invasive quantification of perfusion by using fluorescent dyes, typically Indocyanine Green (ICG). Due to their often leaky and chaotic vasculatures, intravenously administered ICG perfuses through cancerous tissues differently. We investigate here how a few characteristic values derived from the time series of fluorescence can be used in simple machine learning algorithms to distinguish benign lesions from cancers. These features capture the initial uptake of ICG in the colon, its peak fluorescence, and its early wash-out. By using simple, explainable algorithms we demonstrate, in clinical cases, that sensitivity (specificity) rates of over 95% (95%) for cancer classification can be achieved.

An EPR Strategy for Bio-responsive Fluorescence Guided Surgery with Simulation of the Benefit for Imaging.

A successful matching of a PEG group size with the EPR effect for an off-to-on responsive NIR-fluorophore conjugate has been accomplished which allows two distinct in vivo tumor imaging periods, the first being the switch on during the initial tumor uptake via enhanced permeability into the ROI (as background is suppressed) and a second, later, due to enhanced retention within the tumor. Methods: Software simulation (https://mihaitodor.github.io/particle_simulation/index.html), synthetic chemistry, with in vitro and in vivo imaging have been synergistically employed to identify an optimal PEG conjugate of a bio-responsive NIR-AZA fluorophore for in vivo tumor imaging. Results: A bio-responsive NIR-AZA fluorophore conjugated to a 10 kDa PEG group has shown excellent in vivo imaging performance with sustained high tumor to background ratios and peak tumor emission within 24 h. Analysis of fluorescence profiles over 7 days has provided evidence for the EPR effect playing a positive role. Conclusion: Preclinical results show that exploiting the EPR effect by utilizing an optimized PEG substituent on a bio-responsive fluorophore may offer a means for intraoperative tumor margin delineation. The off-to-on responsive nature of the fluorophore makes tumor imaging achievable without waiting for clearance from normal tissue.

Potential for release of pulmonary toxic ketene from vaping pyrolysis of vitamin E acetate.

A combined analytical, theoretical, and experimental study has shown that the vaping of vitamin E acetate has the potential to produce exceptionally toxic ketene gas, which may be a contributing factor to the upsurge in pulmonary injuries associated with using e-cigarette/vaping products. Additionally, the pyrolysis of vitamin E acetate also produces carcinogen alkenes and benzene for which the negative long-term medical effects are well recognized. As temperatures reached in vaping devices can be equivalent to a laboratory pyrolysis apparatus, the potential for unexpected chemistries to take place on individual components within a vape mixture is high. Educational programs to inform of the danger are now required, as public perception has grown that vaping is not harmful.

Synthesis and evaluation of novel chlorophyll a derivatives as potent photosensitizers for photodynamic therapy.

Chlorophyll a exhibits excellent photosensitive activity in photosynthesis. The unstability limited its application as photoensitizer drug in photodynamic therapy. Here a series of novel chlorophyll a degradation products pyropheophorbide-a derivatives were synthesized and evaluated for lung cancer in PDT. These compounds have strong absorption in 660-670 nm with high molar extinction coefficient, and fluorescence emission in 660-675 nm upon excitation with 410-415 nm light. They all have much higher ROS yields than pyropheophorbide-a, and compound 10 was even higher than [3-(1-hexyloxyethyl)]-pyrophoeophorbide a (HPPH). Distinctive phototoxicity was observed in vitro and the inhibition effect was in light dose-dependent and drug dose-dependent style. They can effectively inhibit the growth of lung tumor in vivo. Among them, compound 8 and 11 have outstanding photodynamic anti-tumor effects without obvious skin photo-toxicity, so they can act as new drug candidates for photodynamic therapy.

RGD conjugated cell uptake off to on responsive NIR-AZA fluorophores: applications toward intraoperative fluorescence guided surgery.

The use of NIR-fluorescence imaging to demarcate tumour boundaries for real-time guidance of their surgical resection has a huge untapped potential. However, fluorescence imaging using molecular fluorophores, even with a targeting biomolecule attached, has a major shortcoming of signal interference from non-specific background fluorescence outside the region of interest. This poor selectivity necessitates prolonged time delays to allow clearance of background fluorophore and retention within the tumour prior to image acquisition. In this report, an innovative approach to overcome this issue is described in which cancer targeted off to on bio-responsive NIR-fluorophores are utilised to switch-on first within the tumour. Bio-responsive cRGD, iRGD and PEG conjugates have been synthesised using activated ester/amine or maleimide/thiol couplings to link targeting and fluorophore components. Their off to on emission responses were measured and compared with an always-on non-responsive control with each bio-responsive derivative showing large fluorescence enhancement values. Live cell imaging experiments using metastatic breast cancer cells confirmed in vitro bio-responsive capabilities. An in vivo assessment of MDA-MB 231 tumour imaging performance for bio-responsive and always-on fluorophores was conducted with monitoring of fluorescence distributions over 96 h. As anticipated, the always-on fluorophore gave an immediate, non-specific and very strong emission throughout whereas the bio-responsive derivatives initially displayed very low fluorescence. All three bio-responsive derivatives switched on within tumours at time points consistent with their conjugated targeting groups. cRGD and iRGD conjugates both had effective tumour turn-on in the first hour, though the cRGD derivative had superior specificity for tumour over the iRGD conjugate. The pegylated derivative had similar switch-on characteristics but over a much longer period, taking 9 h before a significant emission was observable from the tumour. Evidence for in vivo active tumour targeting was obtained for the best performing cRGD bio-responsive NIR-AZA derivative from competitive binding studies. Overall, this cRGD-conjugate has the potential to overcome the inherent drawback of targeted always-on fluorophores requiring prolonged clearance times and shows excellent potential for clinical translation for intraoperative use in fluorescence guided tumour resections.

PEGylated BF2-Azadipyrromethene (NIR-AZA) fluorophores, for intraoperative imaging.

Clinical imaging utilising near-infrared fluorescence is growing as an intraoperative aid for the decision-making processes during complex surgical procedures. Existing uses include perfusion assessment and lymph node identification with many new applications currently being proposed and developed. While imaging hardware and software have significantly progressed in recent times, suitable NIR-fluorophores remain a limiting factor. In this report, we describe the design, synthesis, photophysical characterization and in vivo imaging assessment of new PEGylated NIR-fluorophores based on the BF2-azadipyrromethene fluorophore class. The synthetic route includes PEGylation as the final step, thereby allowing routine access to derivatives substituted with different molecular weights of PEG. Absorption and emission wavelength maxima in PBS lie at 690 and 720 nm respectively with quantum yields over 12%. They show excellent photostability and no light induced singlet oxygen production. A time-course of NIR-fluorescence imaging, post i.v. administration, in BALB/c mice showed a rapid and preferential accumulation in the renal excretion pathway within 20 min, indicative of potential clinical usage for intraoperative identification of vial structures along this pathway. Assessment with clinical imaging equipment showed the NIR-AZA fluorophores to be wavelength compatible and brighter than currently used methylene blue (MB), and that they have the ability to be imaged simultaneously with indocyanine green (ICG) offering a potential for dual colour clinical imaging.