The Diffusing Alpha-emitters Radiation Therapy (DaRT) is a novel brachytherapy technique employing 224-radium enriched seeds releasing short-lived alpha-emitting atoms into the tumour. DaRT overcomes the main obstacle in employing alpha radiation for cancer treatments in liquid and solid media caused by their short range. The aim of the study is to improve the DaRT technique with an external radio-opaque template that can help clinicians predict the correct number of sources to achieve tumour coverage. Furthermore, the template is used to aid clinicians in visualizing lesions and their eventual subcutaneous extension. Finally, it is also utilized on treatment day to ensure that the sources are properly inserted into the tumour.The purpose of this study is to discuss how to use an external radio-opaque template in the Diffusing Alpha-emitters Radiation Therapy (DaRT) technique’s pre-planning and treatment stages. This device would help to determine the proper number of sources for tumour coverage, accounting for subcutaneous invasion and augmenting DaRT safety. The procedure will be carried out in a first phase on a phantom and then applied to a clinical case. A typical DaRT procedure workflow comprises steps like tumour measurements and delineation, source number assessment, and therapy administration. As a first step, an adhesive fiberglass mesh (spaced by 2 mm) tape was applied on the skin of the patient and employed as frame of reference. A physician contoured the lesion and marked the entrance points for the needles with a radio opaque ink marker. According to the radio opaque marks and metabolic uptake the clinical target volume was defined, and with a commercial brachytherapy treatment planning system (TPS) it was possible to simulate and adjust the spatial seeds distribution. After the implant procedure a CT was again performed to check the agreement between simulations and seeds positions. With the procedure described above it was possible to simulate a DaRT procedure on a phantom in order to train physicians and subsequently apply the novel approach on patients, outlining the major issues involved in the technique. The present work innovates and supports DaRT technique for the treatment of cutaneous cancers, improving its efficacy and safety. The current standard of care for cutaneous squamous cell skin carcinoma (cSCC) is surgical excision, which involves removing the tumour while leaving a margin of healthy skin. If surgery is not an option or is not viable or ineffective due to tumour resistance, gamma or beta-based external beam radiation and brachytherapy can be used as alternatives ,3,4,5,6.224radium (Ra-224) enriched seeds releasing short-lived alpha-emitting atoms into the tumour [The Diffusing Alpha-emitters Radiation Therapy (DaRT) is a novel brachytherapy technique employing e tumour . The effe tumour ,10. Furte tumour  and an ae tumour .220radon and its progeny, which migrate away from the implant site because of recoil energy and its gaseous nature, for an average distance of 2.5 mm [This technique improves standard gamma-based brachytherapy treatments with the high relative biological effectiveness of alpha particles and higher control of dose to organs at risk in the surrounding area of the implant . Alpha rf 2.5 mm .DaRT is for all intents an interstitial brachytherapy technique, an effective and short procedure, and it is clinically employed for the treatment of cutaneous squamous cell carcinoma (cSCC) at our center Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori”—IRST IRCCS . The technique has recently concluded the “first in man” clinical trial N. CTP-SCC-00 (NCT03015883) with promising results showing a complete response tumour rate of 78.6% and a partial response rate of 21.4% of the lesions . The preTo begin, the treatment will be carried out on a phantom to demonstrate the efficacy of the entire approach as well as the physician training required to safely implant the seeds without the involvement of the patient. Second, the procedure will be demonstrated on a patient to demonstrate its utility in clinical settings.DaRT technique relies on the implantation of 1-cm long Ra-224 loaded seeds directly in the tumour site through needle applicators, as shown in Each seed is loaded with an average activity of 2 µCi and can be ordered in series up to a length of 6 cm. A standard DaRT technique workflow is presented in Given the novelty of the technique we refer to the work by Popovtzer et al.  and ArazA pre implant CT is then performed where the radio opaque trace will be clearly visible together with the insertion points. Using the commercial treatment planning system (TPS) Oncentra Brachy, Elekta, Stockholm, Sweden, contouring of the hypothetical lesion CTV is performed on CT by following the radio-opaque trace and extending it to a depth of 6 mm to simulate a deep lesion. Geometrical planning is finally performed using the TPS software following the radio opaque insertion points. Planned seeds are implanted and the phantom is scanned again on CT. Pre and post implant CT are then fused for coverage verification through MIMmaestro imaging suite . The fusion process is performed employing a bounding box around the lesion and its radio-opaque trace.The technique outlined in the previous paragraph and represented in Following the phantom preparation described in the template-based planning: phantom training section and displayed in In As shown in The mesh tape was placed upon the patch and the skin-drawn lesion reported with the radiopaque ink on the mesh, as shown in The physician now outlines a second CTV in red based on the information from the PET/CT scan about the tumour’s subcutaneous extension. The discrepancy between the visible CTV and the PET/CT based CTV is greater than 6 mm, as shown As a result of these considerations, the required number of sources and seed displacement were updated evaluating the amount of space to fully cover the PET/CT based CTV directly on the radio-opaque mesh, as shown in The DaRT technology recently completed the “first in man” clinical study N. CTP-SCC-00 NCT03015883), and the Food and Drug Administration of the United States awarded it the “Breakthrough Device Designation”. As previously stated by Arazi et al. [83, and tAlpha particles are extremely effective in destroying cancer cells in comparison to standard photon-based techniques, however, their limited range in tissue represent a strong limitation for their application in clinical setting. A requirement to employ particle based radiotherapy is great precision in target localization and dose delivery. An advantage of this technique is that alpha particles are deposited directly into the tumour without the need of large and very expensive technological facilities to accelerate hadrons such for particle external beam radiotherapy. However, precision in target coverage is still a central issue for the DaRT technique. We demonstrated step by step the feasibility of a template based procedure in a phantom and subsequently applied it to a patient, leading to several major benefits in treatment precision and safety.The direct visualization of the lesion on CT via the radio-opaque template will assist physicians and medical physicists in better evaluating lesion extension and, as a result, will provide a better estimation of the number of sources required to achieve full tumour coverage while minimizing needle insertions, as shown in Furthermore, by comparing the naked eye and PET/CT based lesion CTV, millimetric sized subcutaneous lesion extensions that are very difficult to spot can be analyzed. Even increasing the naked eye-based GTV by 6 mm to account for tumour subcutaneous extension and microscopic invasion, the lesion is not totally encompassed by the CTV for another 6 mm, as shown in In The procedure’s flexibility in considering tumour subcutaneous extension and potential growth is the procedure’s last benefit. It is possible to quantify the adjustments required for tumour coverage using the TPS and, as a result, update the template. New tumour contours and needle insertion locations coordinates are derived from the radio-opaque mesh and used to update the physical template, eliminating target misses and optimizing seeds positioning, as shown in The DaRT’s safety and precision were improved in this study, which included a detailed description of physician training on phantom and clinical applications. In particular, when dealing with subcutaneous extension of the tumour, the use of a template may avoid dangerous target miss and ensure complete dose coverage.


Although therapies of cancer are advancing, it remains challenging for therapeutics to reach the sites of metastasis, which accounts for majority of cancer associated death. In this study, we have developed a strategy that guides an anti-programmed cell death-ligand 1 (aPDL1) antibody to accumulate in metastatic lesions to promote anti-tumour immune responses. Briefly, we have developed a combination in which Vadimezan disrupts tumour blood vessels of tumour metastases and facilitates the recruitment and activation of adoptively transferred aPDL1-conjugated platelets. In situ activated platelets generate aPDL1-decorated platelet-derived microparticles (PMP) that diffuse within the tumour and elicit immune responses. The proposed combination increases 10-fold aPDL1 antibody accumulation in lung metastases as compared to the intravenous administration of the antibody and enhances the magnitude of immune responses leading to improved antitumour effects. Cancer metastasis is the leading cause of death in patients, here, the authors show disrupting tumor vasculature could recruit and activate anti-PD-L1 engineered platelet at metastatic tumor sites to block the PD-1/PD-L1 crosstalk and enhance the anticancer immunotherapy. Once a tumour has metastasised, survival is poor, despite treatment with currently available therapies6. Recently, the clinical development of immune checkpoint blockade (ICB) targeting the PD-1/PD-L1 pathway has resulted in unprecedented improvements in overall survival within a broad range of malignancies, however, only a subgroup of patients experience durable benefit10. Challenges that hinder the treatment of metastasis are highly complex and related to an adaptable and heterogeneous biological environment, poor understanding of the molecular and cellular drivers of immune escape, alternations in the cytokine or chemokine milieu affecting cell trafficking to the tumour, tumour-associated organ dysfunction, tumour hypoxia, tumour volume, and drug resistance16. Furthermore, many systemic therapies are associated with toxicities and some can even be prometastatic18. Therefore, a strategy that enhances intratumoural delivery and simultaneously reduces the drug dosages to reach therapeutic effects represents an ideal approach to decreasing toxicity and increasing efficacy.Tumour metastasis remains challenging to eradicate and represents the principal cause of cancer-related mortality20, and multiple strategies have been developed to target the tumour neo-vasculature to treat cancer21. Anti-angiogenesis agents can cause local haemorrhage, activation of the coagulation cascade and platelet recruitment. The latter effect may be exploited to develop targeted drug delivery23. Herein, we report the combinatorial effects of a neo-blood vasculature disruption agent, Vadimezan, and aPDL1-conjugated platelets (P@aPDL1) to promote anti-tumour activity. Vadimezan is a small molecule inhibitor advanced to phase III clinical trials22 that disrupts neo-blood vasculature and induces local tumour bleeding. We hypothesis that platelets loaded with aPDL1 and adoptively transferred can be preferentially recruited to the haemorrhagic tumour site, form platelet-derived microparticles25 and locally release the aPDL1 to enhance T cell-based immunotherapy -cyclohexane-1-carboxylate (Sulfo-SMCC)  was added to prevent platelet activation27. In the 4T1 orthotopic breast tumour model, extensive haemorrhaging occurred 6 h after a single intravenous administration of 15 mg kg−1 Vadimezan as compared to PBS treatment  secretion, which is known to cause haemorrhagic necrosis in tumours22. Vadimezan treatment caused recruitment of platelets within the haemorrhagic tumour and they were activated and identified in the tumour with CLSM  Fig. . The dev+ T lymphocytes in mice with P@aPDL1 and Vadimezan combination treatment as compared with other treatment groups  were 2.7~3.1-fold higher in mice receiving P@aPDL1 and Vadimezan treatment compared to control mice  were detected in mice administrated with P@aPDL1 and Vadimezan  were detected in the tumour, supporting the elicitation of immune responses within the tumour site  were analysed seven days post-treatment , effector memory CD8 T cells (CD8+CD62L−CD44+) and naïve CD8 T cells (CD8+CD62L+CD44−)  in the tumour after the P@aPDL1 and Vadimezan combined treatment compared to other control groups  Fig. . We also30. By inducing haemorrhagic necrosis at the tumour site coupled with engineered platelet recruitment, this approach leverages the physiological interactions of platelet and tumour vasculature to augment targeting capability.To date, a variety of strategies have been advanced to initiate tumour-specific T cell responses in patients, such as ICB, engineered T cells, interferons/interleukins, and tumour vaccines. While these immunotherapeutic approaches are transforming the practice of oncology across a broad spectrum of both solid and hematologic malignancies, response rates are limited, particularly in tumours that lack T cells or an inflamed phenotype. Targeted delivery of the immunotherapeutic agents directly to the metastatic tumours could potentially enhance treatment efficacy as well as reduce side effects32. In our study, we converted platelets into a “Trojan horse” for tumour targeting and loaded with ICB for enhanced tumour killing. Importantly, our proposed strategy aims to develop a method for treatment of already formed metastatic tumours which frequently cause the patient’s death. Moreover, the current therapeutic achievement is based on a one-time treatment, therefore, administration frequency adjustment could further potentiate the efficacy against metastasis.Recent studies show that platelets promote tumour metastasis by physical association and bidirectional activation. Specifically, cloaking protects circulating tumour cells from shear forces and attack by the immune system, while promoting vascular adhesion of tumour cells and its transendothelial migrationκB (nuclear factor κB) signalling pathways to induce robust type I interferon and proinflammatory cytokine responses34. Consistent with the fact that STING activation upregulates PD-L1 expression in tumour cells35, we observed PD-L1 upregulation in 4T1 breast tumours following Vadimezan treatment  is the receptor of Vadimezan, which activates the TBK1/IRF3 (interferon regulatory factor 3) and NF-In summary, we have developed a strategy that combines a tumour vasculature disrupting agent (Vadimezan) with aPDL1-loaded platelets to target tumour metastases. Vadimezan induces tumour vascular endothelial cell apoptosis, which leads to haemorrhagic necrosis at the tumour site. This milieu promotes the trafficking of aPDL1-engineered platelets and their activation to deliver payload aPDL1-PMPs within the tumour site that elicit immune responses. This approach leverages the dynamic role of platelets in human physiology and represents a promising treatment for metastatic cancer.−1 penicillin/streptomycin (Invitrogen) at 37 °C in 5% CO2.Mouse mammary carcinoma cell line 4T1 and mouse melanoma cell line B16F10 and were purchased from the American Type Culture Collection. The B16F10-Luc cell line was purchased from Imanis Life Sciences, the 4T1-Luc cell line and the 4T1-GFP cell line were a gift from Prof. H. Meng at the University of California, Los Angeles (UCLA). Cells were incubated in DMEM  with 10% FBS (Invitrogen) and 100 U mLC57BL/6 and BALB/c mice were purchased from The Jackson Laboratory. Age-matched (6–10 weeks) female mice were used throughout all experiments. All mouse studies were carried out under the protocols approved by the Institutional Animal Care and Use Committee at the University of California, Los Angeles (UCLA).γ , CD44 , CD62L , CD9 , CD41 , CD36 , CD61 , CD62P , CD154 , PD-L1 , CD62P , CD31 , Donkey anti-goat IgG , goat anti-rat IgG . The antibodies were diluted 50-200 times during staining depends on its brightness.The anti-mouse PD-L1 antibody was purchased from BioLegend Inc. . The antibodies used for flow cytometry analysis and immunofluorescence staining including: CD3 , CD4 , CD4 , CD8 , Ki67 , IFN-g, 8 min, 22 °C). Then Prostaglandin E1  was added into the supernatant with a concentration of 1 µM, the supernatant was further centrifuged at 1000 × g for ten minutes to collect the platelets. PBS containing 1 µM PGE1 was used to resuspend the platelets. The platelets number was calculated using a hemocytometer under a microscope. The isolated platelets (1 × 108) were then incubated with Traut’s reagent  (0.1 mg mL−1) for two hours at 22 °C to convert the amine group to thiol group on the surface of platelets. The excess Traut’s Reagent was removed under centrifugation , and further resuspended with PBS with PGE1. In the meantime, the aPDL1 was mixed with sulfosuccinimidyl-4- cyclohexane-1-carboxylate   for 2 h at 4 °C. The unreacted Sulfo-SMCC was removed by centrifugation with a centrifugal filter tube (molecular weight cut-off =10 kDa). Lastly, antibodies were mixed with platelets in PBS containing PGE1 (1 μM). The aPDL1-conjugated platelets were collected with centrifugation . The amount of aPDL1-conjugated to the platelet was determined by ELISA .Platelets were obtained from the blood of mice. Typically, the mouse blood was collected into dipotassium ethylenediaminetetraacetic acid-treated tubes, and the supernatant was obtained with centrifugation of the blood  was added to a glass-bottom 96-well plate and incubated overnight at 4 °C. The native platelets or the P@aPDL1 were resuspended with BSA solution (2%) to block nonspecific interaction (30 min). Then the platelets or P@aPDL1 were stained with Wheat Germ Agglutinin  for 30 min and washed with PBS. The platelets were added to the collagen-coated and non-coated plate (∼1 × 107 cells/well) with 30 s incubation. The platelets binding to the plate were detected under fluorescence microscopy (Nikon) after washed with PBS.For the collagen-binding assay, the collagen solution  and a goat anti-rat IgG (APC-conjugated) for 30 min. After washed by PBS, the P@aPDL1 was observed with a confocal microscope (Zeiss LSM880).g, 22 °C). For thrombin treated P@aPDL1 (PMP) collection, the thrombin treated P@aPDL1 was first centrifuged, then collected supernatant and centrifuged at 15,000 × g for 30 min; then PMP was collected, incubated with 2% uranyl acetate, then mixed with lead citrate for 5 min, and then transferred to a copper grid. The TEM images were obtained with a T12 cryo-electron microscope (FEI Tecnai).P@aPDL1 and thrombin treated P@aPDL1 were fixed in paraformaldehyde solution (4%) for 30 min. Afterwards, the P@aPDL1 was collected by centrifugation , and thrombin with a final concentration of 0.5 U mL−1 was used for stimulation of the platelets. At a different time, 50 μL samples were extracted and further centrifuged for 8 min at 1000 × g. The supernatant containing aPDL1 was collected and the amount of aPDL1 was measured by ELISA kit.Briefly, 1 × 106) to the mammary gland fat pad. PBS or Vadimezan  was intravenously injected when the tumours reached 200 mm3. Six hours post-injection, the mice were sacrificed, the tumours were collected to analyse the haemorrhage and platelet recruitment by immunofluorescence staining.For a study of tumour vasculature disruption after treatment with Vadimezan, an orthotopic breast tumour was established by transplanting 4T1 cells (1 × 105) were injected into the BALB/c mice intravenously. The formation and development of lung metastases were tracked by monitoring the bioluminescence signals with the IVIS, . Eight days later, mice were randomly separated into five groups (n = 8). The mice treated with different formulations, including PBS, aPDL1, Vadimezan, P@aPDL1, and Vadimezan + P@aPDL1 . The bioluminescence signals were recorded on day 10, 15, 20, 25 and 30 after tumour cells inoculation. The Living Image Software was used for the analysis of signals.For a study of treatment efficacy in the metastasis model, luciferase or GFP-tagged 4T1 cells . The mice received treatments with different formulations, including PBS, aPDL1, Vadimezan, P@aPDL1, and Vadimezan + P@aPDL1 . The bioluminescence signals were recorded on day 11, 16, 21 and 26 after tumour cells inoculation.Another mouse melanoma metastatic tumour model was generated by injecting B16F10 cells into the C57BL6 mice intravenously. Briefly, mice were intravenously injected with 5 × 10For observation of tumour growth in the lungs, mice that received different treatments were sacrificed at day 30 (for 4T1 metastasis model) or day 25 (for B16F10 melanoma metastasis model) post-cancer cell inoculation, and the tumour-bearing lung tissues were fixed with paraformaldehyde (4%) solution and stained with H&E to observe the metastasis on the lungs.To study aPDL1 accumulation in metastasis-bearing lung tissues, aPDL1 was labelled by Cyanine5.5 NHS-ester (Lumiprobe) before conjugated to the platelets. After purification, the Cy5.5-labelled aPDL1, P@aPDL1, and Vadimezan + P@aPDL1 were administrated to the mice intravenously (10 days after 4T1 cells inoculation). Twenty-four hours post-administration, lungs were collected, then the aPDL1 in the lungs were detected by IVIS, the fluorescence signals were analysed by Living Image Software.Blood samples were harvested  post-treatment. The cells were stained with antibodies post lysis process to remove the red blood cells, all of the antibodies stainings was followed the manufacturer’s instructions. The stained cells were measured on a BD LSR II flow cytometer with BD FACSDiva 6.1 and analysed with FlowJo 10.7 software.α , IFN- γ , IL-2 , IL-6 , and IL-12  were measured with ELISA 7-day post-treatment. The plasma samples were obtained with centrifugation , and the lung tissue samples were harvested by homogenised in cold PBS buffer in the presence of digestive enzymes and further centrifugated at 2000 × g at 4 °C.The plasma and metastatic site levels of cytokines, including TNF-−1 of anti-mouse CD31  antibody was incubated with the slides overnight, and the donkey anti-goat IgG  secondary antibody was added after three times PBS washing, the nuclei were stained with Hoechst 33342. For platelet staining, 10 μg mL−1 of anti-mouse CD62P  were used. For the T cells staining, 20 μg mL−1 PE anti-mouse CD8a antibody  and Alexa Fluor® 488 anti-mouse CD4 antibody  was used to stain the tumour slices overnight at 4 °C. The tumour cell apoptosis was analysed with TUNEL. The stained slides were measured on a Zeiss LSM880 confocal laser scanning microscope and analysed with Zen 2 and Image J software.Tumours were harvested from the mice and frozen in the cutting medium before sectioning via a cryotome. Before staining, the tissues were incubated with 1% BSA to block the nonspecific binding. For blood vessel staining, 10 μg mLt-test was used for two-group comparisons. The survival benefit was determined using a log-rank (Mantel–Cox) test. All statistical analyses were calculated by Prism (PRISM 7.0). The threshold for statistical significance was P < 0.05. All the experiments were repeated independently with similar results at least three times.All results are presented as the mean ± standard deviation (SD). Tukey post-hoc tests and one-way ANOVA were used for multiple comparisons, and Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


M1 and M3 were found to have a high affinity for imaging the hERG channel in the cell-based experiment. Compared with other fluorescent labeling technologies (such as fluorescent proteins), these probes afford a convenient and economical method to determine hERG channel in vitro and in cellulo. Therefore, these probes are expected to be applicable for usage in physiological and pathological studies of hERG channels and have the potential to establish a screening system for hERG channels.Three environment-sensitive probes were developed for the hERG channel based on the nitrobenzoxadiazole fluorophore herein. After careful evaluation, probes  Therefore, the hERG potassium channel is currently the main anti-target. At the FDA's request, all drugs need to be tested for their affinity for hERG channels to assess their cardiotoxicity (The human ether-a-go-go–related gene (hERG) codes to form a rapidly delayed rectifying potassium ion channel. The channel consists of four identical alpha subunits forming a tetrameric structure and a pore-forming region in the middle. Each subunit consists of six helical transmembrane domains (denoted as S1–S6) . In cardtoxicity .The hERG potassium channel distributes in various cancer cells, neurons, smooth muscle cells of organs, and chromaffin cells, performing different roles in different tissues . Over th2S, H2O2, etc.) directly, or for even tracking dynamic processes in a real-time manner at the animal level . The experimental results display that these probes have rational optical properties . The affinity of probe M3 for the hERG channel is similar to that of M1, with IC50 of 10.8 nM and Ki of 6.08 nM. Although having lower activity than probe M1 and astemizole, probe M2 still displays a potent affinity for the hERG channel , consistent with its affinity from the radioligand binding experiment. The reason why probes M2 and M3 require high concentrations (5 μM) is that the pharmacophore of probes M2 and M3 is E-4031, which has a slightly lower affinity for hERG channels than astemizole. Moreover, a potent hERG channel inhibitor (astemizole) was selected to incubate cells with each probe. After incubation with astemizole, the fluorescence intensity of all probes decreased significantly (In cell imaging, probe ficantly , Table 4M1 and M3 have higher affinity and more vigorous fluorescence intensity than our previously published environment-sensitive probes (Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel, in vivo research.In conclusion, we designed, synthesized, and evaluated three small-molecule fluorescent probes herein. Introduction of the environment-sensitive fluorophore NBD revealed that these probes do not require a time-consuming washing procedure during microscopic imaging. Compared with other reported fluorescent labeling technologies, such as fluorescent proteins, these probes provide a convenient and affordable method for detecting the hERG channel. It should be noted that among all probes, molecules


The study considers the effect of thermophoresis particle deposition on the flow properties of second grade fluid with variable viscosity, variable thermal conductivity and variable concentration diffusivity subjected to a convective boundary condition. To further describe the transport phenomenon, the special case of assisting and opposing flows is explored. Using similarity transformations, the governing equations of the fluid model are transformed and parameterized into a system of nonlinear ordinary differential equations. The approximate analytic solution of a dimensionless system is obtained through the Optimal Homotopy Analysis Method (OHAM). It is observed that velocity and temperature distributions are decreasing functions of the second grade parameter for both assisting and opposing flows. When the thermophoretic parameter is increased, the concentration distributions at the first and fourth orders of chemical reaction decrease. For both opposing and assisting flows, velocity distributions are enhanced due to larger temperature-dependent viscous parameters. The Grashof number is a dimensionless parameter that regulates fluid flow in natural convection and is used in conjunction with heat and mass transfer. This dimensionless number is valuable because it represents the ratio of buoyancy force to fluid viscosity force exerted on a fluid (see1 for details). Furthermore, the Grashof number is crucial because buoyant forces are what causes natural convection as hot and cold fluids flow higher and downward and viscous force tries to halt it. Recently, Koriko et al.2 discussed free convection boundary layer flow of thixotropic fluid with special attention to active and passive controls of nanoparticles. They reported that Grashof number contributes to the augmentation of velocity of the fluid for both active and passive controls of nanoparticles. Likewise, Ramudu et al.3 studied free convective Casson nanofluid flow past a stretching sheet.The mode of heat transfer by which heat energy is transferred between a surface and a moving fluid at different temperature is known as convection. Convective heat transfer is referred to as dominant and special form of heat transfer in gases and liquids mainly because it involves the joint process of heat diffusion and heat transfer by bulk fluid flow. Depending on how the fluid flow is generated, convection can be classified as forced (assisted) or natural (free) convection. An external impetus, such as a fan, pump, or the wind, forces the fluid to flow across the surfaces in forced convection. On the other side convection is termed natural or free if the fluid motion is generated by buoyancy forces that are induced by density difference due to the temperature variation in the fluid. When the fluid is heated from lower layer during the boiling process, thermal expansion occurs and the hotter lower layer of the fluid becomes less dense. Because colder fluid is denser than hotter fluid, buoyancy causes the hotter, less dense part of the fluid to rise while the colder, denser part of the fluid descends and replaces the hotter fluid. When the bottom layer component of the fluid becomes heated and travels upward to be replaced by the cooler fluid, the convective cycle begins. Free convection heat transfer has a wide range of applications. It influences the operating temperatures of power generating and electronic devices, it is important in establishing temperature distributions within the building 15 noticed a particle-free zone surrounding a heated surface in dusty air. Aitken16 demonstrated that the effect was caused by a higher bombardment of particles from molecules in the heated zone compared to the cool region. The gas molecules migrating from the hot side of the particles have a higher velocity than those migrating from the cold side in this event. The particles clash with the molecules more strongly as they move quicker. According to Hayat and Qasim17, this phenomena has several applications in aerosol technology, silicon thin film deposition, and radioactive particle deposition in nuclear reactor safety simulations. Therefore, the velocity of particles is called thermophoretic velocity and the force experienced by suspended particles owing to the temperature gradient is called thermophoretic force with the force's direction opposing the temperature gradient (Stanford18). Recently characteristics of thermophoresis and Brownian motion on radiative reactive micropolar fluid was explored by Mabood et al.7.Thermophoresis of particles is defined as the movement of tiny particles in the direction of a diminishing temperature gradient. In other words, particles in a heated environment move to a colder environment during the process. Tyndal19. The order of chemical reactions is known to be influenced by a variety of factors, the most basic of which is the first-order reaction, in which the rate of reaction is proportional to the concentration of species, Kundu et al.20. According to Themelis21, the majority of chemical reactions represented in applications are first-order when the reaction rate is dependent on a single component and the exponent value is one. Rahman and Uddin22 presented boundary layer flow of nanofluid with variable chemical reaction in a radiative vertical plate. Effects of thermal slip and chemical reaction on free convective nanofluid from a horizontal plate was investigated by Alsenafi and Ferdows23. Panigrahi et al.24 discussed impact of chemical reaction on Mhd flow between vertical walls. Ferdows and Al-Mdallal25 investigated the impact of chemical reaction on boundary layer flow with heat and mass transfer over a linearly stretched sheet. They reported that when the order of the chemical reaction rises, the flow profiles increase, and as the Schmidt number and chemical reaction parameter increase, the flow profiles decline.A chemical reaction occurs when one or more substances are transformed into other ones. Chemical reactions are an essential part of both technology and daily living. Chemical reaction-based processes include the combustion of fuels, the production of glass, the brewing of beer, and the production of wine. In many cases, the rate of chemical reactions is determined by the concentration of the species itself, and the order of chemical reactions with respect to a given substance  is known as the index or exponent to which the concentration term in the rate equation is raised, McNaught and Wilkinson26 studied bioconvection flow of non-Newtonian fluid embedded in porous medium with variable properties. Magnetized nanofluid flow of ferromagnetic nanoparticles from parallel stretchable rotating disk with fluid properties is explored by Shamshiddin and Mohamed27. Ferdows et al.6 deliberated a free convective power-law with variable viscosity and thermal conductivity. Mhd non-Newtonian fluid flow past an exponentially stretching surface with variable thermal conductivity was investigated by Anantha et al.28. Variable thermal conductivity and heat source/sink effects on mhd boundary layer flow past a linearly stretching sheet was studied by Sharma and Singh29. Omowaye and Animasaun30 reported upper-convected Maxwell fluid flow over a melting surface in a hot environment subject to thermal stratification with variable thermo-physical properties.It is a reality that a little rise in temperature boosts transport phenomena by lowering viscosity across the momentum boundary layer, therefore altering the heat transfer rate at the wall. The fluid properties that are more responsive to a rise in temperature are viscosity and thermal conductivity. Nima et al.The current work considers the analysis of the flow of a chemically reacting boundary layer fluid in a thermophoretic second-grade fluid with variable viscosity, variable thermal conductivity, and variable concentration diffusivity embedded in a porous medium. The approximate analytic solution is obtained through Optimal Homotopy Analysis Method. The effects of embedded flow controlling parameters on fluid velocity, temperature, concentration, and shear stress were shown and analyzed graphically.31, Dunn and Fosdick32. The constitutive law of incompressible homogeneous fluids of degree 2 is given byThe introduction of Second grade fluid is to illustrate certain nonlinear effects that cannot be explained by the classical theory of Navier–Stokes for Newtonian fluids  as,Here, Equations , 7), ,  are subjThe continuity Eq.  is satisThe Eqs. , 9), , , 16) an an16) anSubject toThe momentum, energy and concentration equations can be transformed into corresponding ordinary differential equations by the following transformationsThus, tituting  into Eqstituting –23, we oThe corresponding boundary conditions take the formIn the equations above Invoking the rule of solution expressions above for ϕo(η) on –27 togetϕo(η) on  and 29)\documentnditions  and (29)Linear operators The operators In which If Subject to boundary conditionsand By Taylors series expansion one has;Clearly, the convergence of series Eqs. –37 is clies Eqs. –37 conveIf we denote the special solution 42 we choose auxiliary functions asHere,  of Eqs. –49\documIt is obvious that the series –46 consiComputations were conducted using the approximate analytic method described above for various values of the second grade parameter mentclass2pt{minimmentclasspt{minimaIn Fig. Likewise, from Fig. Figure mentclasspt{minimaIt is observed that the magnitude of the concentration profile decreases with larger values of Likewise, the same effect is observed in Fig. Velocity and temperature profiles are decreasing functions of second grade parameter Within the boundary layer, velocity profile is an increasing function of temperature-dependent viscous parameter Both velocity and temperature profiles are increasing functions of temperature dependent thermal conductivity parameter The velocity and concentration profiles increase with increase in concentration dependent diffusivity parameter For first and higher orders of chemical reaction, the magnitude of the concentration profile decreases with larger values of thermophoretic parameter The study of the variable thermophysical properties of second-grade fluid flow past a vertical surface with convective boundary condition with special attention to assisting and opposing flows has been studied analytically. The effects of thermophoresis, temperature-dependent viscosity, temperature-dependent thermal conductivity, and variable concentration diffusivity are all taken into account. Using similarity transformation, the governing partial differential equations are converted into nonlinear ordinary differential equations. This investigation yielded the following significant findings:


Behavioral characteristics of living organisms may affect the metabolism and its underlying molecular basis. The lifestyles of some modern communities include prolonged light exposure at night, and a high-fat/high-sugar-containing diet is frequently investigated. The molecular mechanisms of this unhealthy behavior might involve Adropin and some related nuclear receptors. This study examines the effect of long-term continuous light exposure and high fat/sucrose (HFS) diet on Adropin expression, RORα, Rev-erb-α nuclear receptors, key enzymes of lipid metabolism, and energy homeostasis in a rat model. The results of this study demonstrate the deleterious effects of this modern behavior on the metabolism, histology of many organs and general health. In conclusion CL and WD produced widespread derangements of energy homeostasis and lipid metabolism. The pathogenesis of this disturbance includes, at least in part, the Adropin hormone with the involvement of the RORα and Rev-erb-α nuclear receptors. Future therapeutic potential may involve Adropin. n = 8/group): (a) Normal control group (NC), (b) CL group, (c) WD group, and (d) CL + WD group. Then, indirect calorimetry and food intake (FI) were measured. Finally, Adropin, hormone-sensitive lipase (HSL), adipocyte triglyceride lipase (ATGL), and free fatty acids (FFA) were measured. Additionally, the histopathology and gene expression of Enho, RORα, and Rev-erb-α genes were done. The CL alone elevated the Adropin plasma level and gene expression, increased RORα expression, and decreased the Rev-erb-α nuclear receptor expression mainly in the liver and kidney. Besides, CL increased the total energy expenditure (TEE) and decreased the respiratory quotient. WD alone or in combination with the CL reversed gene expression of Enho, RORα, and Rev-erb-α. Combined CL and WD increased the TEE, reduced the food intake, increased the ATGL, and reduced the Adropin level in addition to widespread degenerative changes in the liver, spleen, and renal tissues. The deleterious effects of CL and WD on energy homeostasis may include Adropin with the involvement of the RORα and Rev-erb-α nuclear receptors.Long-term continuous light exposure (CL) and western diet (WD) effects on Adropin expression, RORα, and Rev-erb-α nuclear receptors and energy homeostasis were studied in rats. Thirty-two male Wistar rats (250–290 g) were enrolled for 3 months in the following groups ( The chronobiology is highly sensitive to changes in the light/dark cycle . Light sSeveral reports stated an emerging role of Adropin as a new player in chronobiology . AdropinConsidering the above data, little is known about the effects of the modern lifestyle with extended periods of light exposure and consumption of high fat and added sugars on the Adropin system and the coupled nuclear receptor. Therefore, this study aimed to investigate the effect of long-term continuous light exposure and a Western diet consisting of high fat/added sucrose (WD) on the Adropin expression, expression of RORα, Rev-erb-α nuclear receptors, key enzymes of lipid metabolism, and energy homeostasis in a rat model.n = 8/group) for 3 months of intervention. The groups included: (a) Normal control group (NC) with normal light/dark cycle, where the light was switched on at 6 a.m. and turned off at 6 p.m., and also fed a balanced diet ; (b) Continuous light (CL) group, in which rats were also fed the balanced diet (the same as that of the NC group) and exposed to continued light exposure for three months [In total, thirty-two male Wistar rats  were obtained from the animal house of College of Pharmacy, King Saud University. Rats were randomly enrolled in one out of four groups 2), volumes of carbon dioxide (VCO2), and total energy expenditure (TEE) per hour per Kilogram (kg) of rat body weights per kg of lean body mass (0.75% of body weight) and per rat weights were done. Besides, the respiratory exchange ratio (RQ), automatic food intake (FI), and manual water intake were recorded [All rats were individually housed in Calo-cages of the PhenoMaster system  at a normal room temperature (25 °C). The first 6 h of measurement were deleted for compensation of acclimatization bias . For thrrecorded . During After indirect calorimetry, 3 rats from each group were randomly selected and operated to insert a carotid catheter  under geImmediately after the cardiac puncture, tissue samples from the liver, kidney, and spleen were collected in blank tubes and frozen immediately in liquid nitrogen and stored at −80°C for gene expression analysis, and another set of tissue sample were fixed in 10% neutral formalin solution for the pathology examination. Then, pathology samples were preserved in 70% ethanol and dehydrated with ascending grades of ethanol. Then, it was cleared with xylene and embedded in paraffin wax to get paraffin blocks. Paraffin blocks were cut to obtain serial sections of 5 μm; then, they were put on the top of glass slides and stained with hematoxylin and eosin (H&E) staining. They were examined under a light microscope equipped with a high-resolution digital camera (×200 and ×400). Three rats from each group were used for pathology, and for each rat, two slides from each organ were stained. The regions of interest (ROIs) were randomly selected and viewed under the microscope. Digital images of histological sections were analyzed using image J software , . Total RNA was isolated from frozen liver tissue according to the RNA isolation kit  following the manufacturer’s protocols. The purity and integrity of the total RNA were monitored by absorbance of an ultraviolet spectrophotometer at 260/280 nm. For the synthesis of complementary DNA (cDNA), the extracted RNA was reverse-transcribed by QuantiTect SYBR Green RT–PCR kit , as recommended by the manufacturer. The primers were designed with Primer3 software and are listed in Plasma Adropin levels were measured using enzyme-linked immunosorbent assay (ELISA) kits for rat Adropin , as described by the manufacturer’s catalog. The key enzymes of the lipid metabolism, such as Hormone-sensitive lipase (HSL) and adipocyte triglyceride lipase (ATGL), were measured in the plasma samples by ELISA Kits,  with the sensitivities of <2 pg/mL and 0.469 ng/mL, respectively. The free fatty acid (FFA) concentration was assessed by kits for the FFA quantification colorimetric assay .p-values < 0.05 were statistically significant. SPSS, version 25 for Windows software  was used for the statistical analyses.The data were presented as the means ± standard deviation (SD). Shapiro–Wilk test was used to test the normality of study variables. Analysis of variance (ANOVA) with an LCD post hoc test was used to analyze the differences in multiple comparisons; additionally, the significance in the multiple comparisons were tested by Bonferroni correction. p < 0.001) (p = 0.173) (p > 0.05). Although both HSL and ATGL were insignificantly changed (p > 0.05), the level of the FFA was significantly reduced (might be cleared for oxidation). In comparison to the NC group, a histopathology examination of the liver showed congested hemorrhagic areas of different sizes and surrounded by degenerated hepatocytes  . The pea< 0.001) . Besides= 0.173) . Compareatocytes A,B. Sectatocytes CL. SectiCompared to the NC group, a Western diet alone significantly reduced the Adropin gene expression in the studied peripheral tissues and decreased its plasma level. Furthermore, the expression of RORα was parallel to the Enho gene, while Rev-erb-α showed a high expression . Changesp-value of the post hoc test = 0.058)  . FurtherThe patterns of the peripheral circadian changes in plasma Adropin were presented in The current study investigated the individual and combined effects of two factors related to the modern lifestyle on metabolic health. The first factor was the extended exposure of light at the dark phase of the normal light/dark cycle (CL), and the second was the ingestion of foods with a high-fat proportion and added sugars containing beverages (WD). The effects on energy homeostasis, lipolysis key enzymes, Adropin peptide, and related nuclear receptors will be discussed here.In the CL group, Adropin expressions in peripheral tissues, i.e., liver, spleen, and kidney, were increased, and the plasma level was elevated. This effect was associated with a significant reduction of the free fatty acid level in the plasma, with insignificant changes in the lipolysis key enzymes (HSL and ATGL), meaning that Adropin may have an independent antilipolytic effect. In-line with this assumption, Gao et al.  stated tBesides Adropin, the sympathetic nervous system (SNS) is also disturbed by extended periods of light exposure. Light/dark cycle alterations could modify the norepinephrine (NE) level in tissues innervated by SNS directly by actions on the rostral ventrolateral medulla, superior cervical sympathetic ganglia, and alterations in the hormonal release, resulting in sympathetic hyperactivity . The hypFurthermore, CL could disturb other modulators of the metabolism, such as melatonin  and glucThe deleterious effect of the CL on the liver, spleen, and renal histology could support the theory of SNS overactivity . The widespread degenerative changes in the studied tissues were like pathological findings in stress models, which were created in experimental animals by the chronic administration of epinephrine for four weeks  or foot Rats fed a high-fat diet with added sucrose (in the WD group) for 12 weeks failed to produce a significant weight gain. Going through the indirect calorimetry study, it was evident that the TEE and RQ were insignificantly changed when compared to the NC group. Moreover, the automatic measurement of the food intake revealed a significant reduction in the food intake in the WD group. In contrary to these findings, Collins et al.  found thRats in the WD group showed significantly reduced the Adropin gene expression and plasma level together with the under-expression of RORα and overexpression of Rev-erb-α. Many previous reports associated the reduction of Adropin levels with insulin resistance conditions. A low Adropin level can be considered a risk factor for insulin resistance and other components of the metabolic syndrome . AdditioThe theory of insulin resistance was clear in the histopathological changes in the liver parenchyma, where a picture like nonalcoholic fatty liver disease (NAFLD) was evident with early to advanced widespread steatosis in all studied slides of the WD group. It was reported that a low serum level of Adropin was associated with NAFLD in adult patients. The hypoadropinemia was linked to the NAFLD pathogenesis through Adropin-related endothelial dysfunction, depending on the ability of Adropin to induce the expression of nitric oxide (NO) into the endothelium, i.e., hypoadropinemia will be associated with a reduction of the NO in endothelial cells . Sterol The combined effect of CL and WD indicated that the WD effect on Adropin is more potent than CL, i.e., Adropin level and expression together with expressions of RORα and Rev-erb-α were like the case in the WD. On the other hand, the stressing effect of adding CL to WD (state of insulin resistance) was synergistic regarding the indirect calorimetry findings  and degenerative changes in the studied tissues. Regarding the energy homeostasis, long-term combined CL and WD further increased the TEE and lowered RQ (toward fat utilization) and reduced the food intake, blunting the weight gain. The finding was corresponding to a previous study showing that CL immediately increased the weight gain then stabilized or even reduced it later due to loss of the central circadian rhythm of energy expenditure and feeding as a result of low amplitude firing of the SCN, whereas the weight gaining effect by the traditional high-fat diet became evident at a later stage than that by the CL, indicating that the CL has an independent mechanism of wight affection .The plasma Adropin circadian rhythm in the NC showed small peaks at night that might be attributed to the time of feeding . On the The current study encountered some limitations, such as a lack of measurement of the other indicators of insulin resistance. Alternatively, we depended on the FFA level and dysregulated HSL/ATGL balance as indicators of insulin resistance. Additionally, we measured the TEE rather than the resting energy expenditure. However, the effect of the TEE in energy homeostasis is the best. Another limitation is the lack of measurement of stress indicators or signs of SNS activation. During the study design, it was not clear that the CL will behave as a stress stimulus, and our objective was directed mainly toward Adropin and its associated nuclear factors. Additionally, the comparison with brain and adipose tissue parameters was missing.In conclusion, the widespread deleterious effects of the CL and WD on the energy homeostasis and lipid metabolism may include the Adropin peptide with the involvement of the RORα and Rev-erb-α nuclear receptors as a mechanism. The effect of WD was more potent than CL regarding the Adropin expression in peripheral tissues and the plasma level, while the degenerative histological changes showed a synergistic effect of both CL as a stressful factor and WD as an insulin resistance condition.


Cancer associated fibroblasts (CAFs) are a major component of the tumour microenvironment in most tumours, and are key mediators of the response to tissue damage caused by tumour growth and invasion, contributing to the observation that tumours behave as ‘wounds that do not heal’. CAFs have been shown to play a supporting role in all stages of tumour progression, and this is dependent on the highly secretory phenotype CAFs develop upon activation, of which extracellular matrix (ECM) production is a key element. A collagen rich, stromal ECM has been shown to influence tumour growth and metastasis, exclude immune cells and impede drug delivery, and is associated with poor prognosis in many cancers. CAFs also extensively remodel their metabolism to support cancer cells, however, it is becoming clear that metabolic rewiring also supports intrinsic functions of activated fibroblasts, such as increased ECM production. In this review, we summarise how fibroblasts metabolically regulate ECM production, focussing on collagen production, at the transcriptional, translational and post-translational level, and discuss how this can provide possible strategies for effectively targeting CAF activation and formation of a tumour-promoting stroma. Fibroblasts are one of the most abundant cell types in the microenvironment of solid tumours, and have long been known to play multiple and varied roles in promoting tumour progression and metastasis. Fibroblasts are influenced by tumour cells to become ‘activated’, a process during which they develop a highly secretory phenotype involving production of growth factors, pro-angiogenic factors, immunomodulatory factors, metabolites, extracellular vesicles, and, crucially, ECM components and remodelling factors –5. ActivIn vivo, the effects of the ECM on tumour growth have been assessed in several studies. Ablation of Col6a1 or Col5a3 in the MMTV-PyMT mammary tumour model resulted in reduced hyperplasia and primary tumour growth (tm1jae) showed increased tumour growth in the MMTV-PyMT model , of which collagen proteins are one of the most highly abundant components, and indeed comprise approximately 30% of the total protein content of mammals . Upon fir growth , 27. ConMT model , 28. InhMT model , 30. TheMT model –33.via conversion to glutamate, or from arginine via conversion to ornithine. Synthesis of 1 mole proline from 1 mole glutamine or arginine requires 8 or 2.5 moles ATP, respectively  , two stu-workers  also fou-workers . These s-workers , includivia ALDH18A1 or OAT), which is the final precursor for proline synthesis by PYCR1, PYCR2 or PYCR3. It seems clear that proline synthesis is upregulated in activated fibroblasts and a limiting factor in collagen production. Hepatic stellate cells increase proline production from glutamine upon activation during liver fibrosis, and PYCR1 is upregulated and proline oxidase (PRODH), which recycles proline back to P5C, is downregulated, showing that fibroblast activation pushes cells towards proline synthesis, although whether this affected collagen production was not investigated   and Hif1a. Interestingly, the authors also found that glutamine depletion post-TGFβ treatment did not affect αSMA protein levels but did affect the other markers, and that α-ketoglutarate only restored Acta2 and Hif1a expression under glutamine deprivation , which has been shown in several studies to inhibit fibrosis and collagen production by fibroblasts –106. TheTGFβ is well-known as a master regulator of CAF activation, but it is becoming clear that it is also a major architect of metabolic rewiring in fibroblasts. TGFβ stimulates glycolysis, serine and glycine metabolism, glutamine metabolism, and increased proline synthesis from glutamine and arginine. Therefore, in addition to increasing ECM gene expression, TGFβ also activates metabolic pathways that support ECM production by activated fibroblasts: by increasing ATP generation to support synthesis of ECM protein, increasing production of amino acids required for collagen translation and by enhancing collagen stability and post-translational modification.in vivo, since the majority of research to date has focussed on the role of these pathways in 2D cell culture of activated fibroblasts and the only in vivo models have been of wound healing or fibrotic disease.Glycine, proline, glutamine and arginine metabolism are all potential targets for normalising collagen production in the tumour stroma to reduce tumour growth and improve tumour perfusion and drug delivery. However, there is still much research to be done and many unanswered questions. Firstly, many of the studies showing these metabolites affect collagen production have been carried out in the context of activated fibroblasts in wound healing, fibrotic disease and acutely TGFβ-treated fibroblasts, rather than CAFs derived from cancer patients, although gene expression data suggests these pathways are also upregulated in the tumour stroma. Therefore, further research is needed to verify that metabolic changes in CAFs are relevant and targetable pathways to regulate ECM production. Furthermore, there is a lack of studies investigating whether these metabolic pathways could be a viable therapeutic target against the tumour stroma KRas-induced PDAC mouse model, ablation of stromal collagen I enabled recruitment of tumour suppressing myeloid cells and promoted tumour progression  and Breast Cancer Now (2019AugPR1307 to SZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The authors regret that the funding information was incorrectly shown in the acknowledgements section of the original manuscript. The corrected funding acknowledgement is as shown below.This work was supported by Swedish research counsel VR (grant 2016-05904), Swedish research counsel FORMAS (grant 2016-00908), the Estonian Research Council grant PRG627, and the Estonian Centre of Excellence project TK141.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


E. coli (EC) causing UTI in U.S. patients and the activity of TBP and comparators against various subsets.Tebipenem (TBP) is an oral carbapenem in clinical development for treating complicated urinary tract infections (UTIs), including pyelonephritis. This study investigates the epidemiology of A total of 2,395 EC recovered from urine samples during the 2018-2020 STEWARD Surveillance Program were included. Isolates were collected from medical centers in all 9 US Census Regions and centrally tested by reference broth microdilution method. MIC interpretation was based on CLSI criteria. Isolates that met MIC criteria were subjected to genome sequencing, followed by screening of extended-spectrum β-lactamase (ESBL) genes and epidemiology typing (MLST).blaCTX-M and 2/360 (0.6%) had blaSHV-12. blaCMY  was the most common cephalosporinase, followed by blaDHA . A CRE phenotype was noted in 1 isolate from New York, which carried blaKPC-2. Acquired genes were not detected in 56 strains. 50 ST types were noted in isolates that met the ESBL criteria screening, with the majority of isolates being ST131 (56.2%). 21 (6.7%) and 19 (6.0%) isolates belonged to ST38 and ST1193, respectively, followed by STs represented by 8 or less isolates. Among ST131, 56.5% carried blaCTX-M from group 1 and 35.6% had genes associated with group 9. Overall, TBP showed consistent MIC50 values throughout the subsets. ERT had activity (≥97.0% susceptible) against the various subsets; however, lower susceptibility rates (85.7-90.6%) were noted against isolates carrying plasmid AmpC. Other agents (ceftriaxone and cefazolin) had activity only against non-ESBL producers.A total of 16.1%, 15.4% and 14.6% of EC met the ESBL screening criteria in 2018, 2019 and 2020, respectively. 269/360 (74.7%) carried blaCTX-M comprised the majority of acquired genes detected among ESBL strains, which belonged mostly to ST131, emphasizing the expansion of this clone. TBP showed consistent activity against all subsets, regardless of resistance genotype or lineage. These data support the clinical development of TBP as a convenient oral treatment option for UTI caused by EC.Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Timothy B. Doyle, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Ian A. Critchley, Ph.D., Spero Therapeutics  Nicole Cotroneo, Spero Therapeutics  Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  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S-Transferases (GSTs), including NI3 which we developed recently based on 1,8-naphthalimide (NI), are limited by their short emission wavelengths due to insufficient penetration. To realize imaging at a deeper depth, near-infrared (NIR) fluorescent probes are required. Here we report for the first time the designing of NIR fluorescent probes for GSTs by employing the NIR fluorophore HCy which possesses a higher brightness, hydrophilicity and electron-deficiency relative to NI. Intriguingly, with the same receptor unit, the HCy-based probe is always more reactive towards glutathione than the NI-based one, regardless of the specific chemical structure of the receptor unit. This was proved to result from the higher electron-deficiency of HCy instead of its higher hydrophilicity based on a comprehensive analysis. Further, with caging of the autofluorescence being crucial and more difficult to achieve via photoinduced electron transfer (PET) for a NIR probe, the quenching mechanism of HCy-based probes was proved to be PET for the first time with femtosecond transient absorption and theoretical calculations. Thus, HCy2 and HCy9, which employ receptor units less reactive than the one adopted in NI3, turned out to be the most appropriate NIR probes with high-sensitivity and little nonenzymatic background noise. They were then successfully applied to detecting GST in cells, tissues and tumor xenografts in vivo. Additionally, unlike HCy2 with a broad isoenzyme selectivity, HCy9 is specific for GSTA1-1, which is attributed to its lower reactivity and the higher effectiveness of GSTA1-1 in stabilizing the active intermediate via H-bonds based on docking simulations.The applications of most fluorescent probes available for Glutathione  An abnormal and intriguing phenomenon that the fluorophore's electron-deficiency could affect a probe's performance is now revealed for the first time. S-Transferases , GSTA, GSTM and GSTP are regularly overexpressed in tumor cells, especially in those with drug-resistance.1–5 With little toxicity, high sensitivity, a fast and convenient detection process, and facile modification, more and more attention has been drawn to small molecule fluorescent probes.6–12 Recently, with the 1,8-naphthalimide (NI) scaffold, we have developed a two-photon fluorescent probe NI3 for GSTs after an elaborate investigation into the structure–activity relationships between nonenzymatic or enzymatic reactivities and the effective electrophilicity of the receptor unit.13 Despite NI's two-photon absorptivity, its short emission wavelength impeded more practical applications such as in vivo imaging. It would be desirable that both excitation and emission wavelengths are in the near-infrared (NIR) region (650–900 nm) due to deeper skin and tissue penetration, lower background autofluorescence interference, and less phototoxicity.14–19 In addition, NIR fluorophores usually possess a higher molar extinction coefficient ε, and as can be seen in Three subclasses of cytosolic Glutathione E)-2-vinyl)-3,3-dimethyl-1-ethyl-3H-indol-1-ium iodide (HCy) was selected as the fluorophore in light of its NIR character, high brightness (εφ) and favorable photostability.20,21 What's more, a systematic study on whether and how replacement of the fluorophore could affect a probe's recognition behavior is lacking. Herein, to address this very issue and to develop highly sensitive NIR fluorescent probes for GSTs with negligible background noise for the first time, receptor units adopted in NI-based probes were linked with HCy to produce the NIR probes, which unexpectedly appeared to be more reactive than NI-based ones. Subsequent studies on the hydrophilicity and the electron-deficiency of the fluorophore demonstrated that the latter is the origin of the higher reactivity. Meanwhile, the fluorescence caging mechanism of HCy-based probes was inspected with femtosecond transient absorption spectra and theoretical calculations. Thus, instead of 4-cyano-2-nitro-benzenesulfonyl adopted in NI3, HCy2 and HCy9 with more inert receptor units turned out to be the most appropriate NIR probes for practical applications, including GST imaging in cells, tissues and even tumor xenografts in nude mice. In addition, the different isoenzyme selectivities of these two probes and the specificity of HCy9 for GSTA1-1 were discussed according to the docking simulations.Hence,  could significantly suppresse the fluorescence increase by inhibiting these activities. Subsequent selectivity experiments illustrated the probe's specificity for GST activity  of HCy (28 440 M−1 cm−1)20 relative to NI's (660 M−1 cm−1),25 which equivalently means amplifying the signal of a certain reaction behavior. The other one is the higher reactivity (knonc) of HCy-based probes. To identify if the second one hits the bull's eye, kinetic study on nonenzymatic reactions was conducted. As shown in HCy-based probes are indeed more prone to reacting with GSH than NI-based ones, regardless of the specific receptor unit. These results demonstrated that a probe's reactivity is not only related to the receptor unit but also to the fluorophore. So, when regarding the reactivity, what causes the difference between HCy and NI?Interestingly, although the detection mechanism doesn't change with the replacement of the fluorophore from d Fig. S3. Notably Fig. S3j, which dHCy due to its net positive charge, which is conducive to encountering similarly hydrophilic GSH molecules. The second one is the higher electron-deficiency of HCy due to the same positive charge, which favors the nucleophilic attack of GSH on the electrophilic center via the electronic effect and maybe electrostatic attraction (GSH bearing negative charges) as well. To discern which one accounts for this phenomenon, several parameters based on rate constants knonc, octanol–water partition coefficients log P and the local electrophilicity ωk, respectively, were defined and inspected. Therein, the log P values of HCy-based probes calculated with the well-known ALOGPS 2.1 program26–28 are truly smaller irrespective of the specific receptor unit, indicating their higher hydrophilicity  the receptor unit is, the less the enlargement factor of ωk (i.e. R4) or knonc (i.e. R1) is, could be interpreted and comprehended readily as follows. In a probe molecule, just as demonstrated above, either a more electrophilic receptor unit or a more electron-deficient fluorophore will induce its higher reactivity with GSH. Hence, when a probe is endowed with a more electrophilic receptor unit, the additional elecrtrophilic effect brought about by the introduction of a more electron-deficient fluorophore seems not that notable, sort of like the law of diminishing marginal utility in economics  implies that the stronger enzymatic reactivity of HCy-based probes stems from their stronger nonenzymatic reactivity. Besides, the Km values of HCy-based probes are universally smaller than those of NI-based ones,13 indicating their stronger binding ability with GSTs. Consequently, the larger kcat and smaller Km make HCy-based probes more prone to recognizing and detecting GSTs to the extent that the catalytic efficiency kcat/Km of HCy9 for GSTA1-1 is 0.124 ± 0.021 s−1 μM−1, a value comparable to that of NI3 (0.103 ± 0.011 s−1 μM−1),13 the receptor unit of which is a more electrophilic one, implying HCy9's practical detection ability for GSTA in cells (vide infra). Another noteworthy result is that the susceptibility of knonc (6.44) towards ωk is still larger than that of kcat (0.72)  Fig. S9, analogoe Fig. S6 showed Hvia photoinduced electron transfer (PET) due to the relatively smaller excitation energy, which results in the failure to produce charge separation.31–36 In other words, NIR fluorophores usually have a lower LUMO and/or higher HOMO, and this will reduce the driving force (ΔGPET) of the PET process regardless of the donor-type (d-PET) or acceptor-type (a-PET)  relative to NI9.13 Additionally, as an isomer of HCy10, HCy9 was found to exhibit obviously stronger autofluorescence than the former . Consequently, we examined if the quenching mechanism of HCy9 was still PET and explored the specific inherent reason for the above phenomena. For this purpose, with HCy as the control molecule, measurements of the femtosecond transient absorption (TA) spectra and calculations of electronic transitions based on time-dependent density functional theory (TD-DFT) were performed. Upon excitation using a laser at 630 nm, a distinct transient absorption band of HCy centered at ca. 535 nm appeared instantly  signal centered at ca. 535 nm was observed in the first 60 fs upon excitation at 630 nm  , which can be populated directly from the S0 state upon excitation since the f of the corresponding transition is considerable (1.274). Noticeably, the S0 → S2 transition corresponds to HOMO → LUMO+1  state. Hence, the transformation from S2 to S1 is essentially the PET process, and the new state generated in the second 60 fs corresponds to the CT state (1 of HCy can be populated directly from the S0 state (f = 1.124) 13 arising from the lower LUMO of the NIR fluorophore (−3.0 eV vs. −2.7 eV).13 And the higher quenching efficiency of HCy10 relative to HCy9 is probably related to the lower LUMO of the quencher moiety (i.e. the receptor unit)  of the receptor unit can also affect the probe's fluorescence behavior.It should be noted that relative to non-NIR probes, NIR ones are generally more difficult to regulate Scheme S2. This ca<100 fs; , which s<100 fs; . As for t 630 nm  and S10†. 460 nm , indicaty moiety , left, dCT state . As a co Table S8, right, 13 10 μM HCy2 or HCy9 in HEPES buffer  was used to incubate HepG2 cells, after which fluorescence images were captured. As shown in Fig. S12, S13 and Video in the ESI,N-ethylmaleimide (NEM), respectively. For either HCy2 or HCy9, both EA and NEM could induce an obvious fluorescence loss  for HCy9 or HCy2 into these three GST isoenzymes were implemented. For the GS-HCy9 σ complex, in regard to GSTA1-1, abundant H-bonds are formed between residues in the active-site and the receptor unit moiety of HCy9  of a probe is related to both the receptor unit and the fluorophore. Specifically, the higher the electron-deficiency of the fluorophore is, the more prone to reacting with GSH the probe is, which makes There are no conflicts to declare.SC-011-D0SC04411C-s001SC-011-D0SC04411C-s002


The Department of Defense Grant award that helped support this work was W81XWH-16-1-0025. Therefore, he needed to change financial support and sponsorship in his publication in our journal.According to the evidence provided by the corresponding author, this work was supported by the National Cancer Institute Grant (R01CA200232-05) and the Department of Defense Grant (W81XWH-16-1-0025) both to Maki CG, and all of the other authors of this paper agreed with the change mentioned above. Therefore, we publish this erratum to announce this change.Cancer Drug Resist 2021;4:85-95. http://dx.doi.org/10.20517/cdr.2020.85Cite this article: Shim D, Duan L, Maki CG. P53-regulated autophagy and its impact on drug resistance and cell fate.


Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable ‘turn-on’ fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules. The rise of fluorescence spectroscopy as a means to visualize biological systems at the molecular level has led to substantial findings about cellular and physiological mechanisms. Fluorescent probes have been heavily employed to monitor and report on a variety of processes such as the dynamics of cellular structures, protein expression and localization, enzymatic activity, and regulation of signaling networks ,2. Such Examples of such advancements are ‘turn-on’ or ‘turn-off’ fluorescent probes, which can change their fluorescent output after interacting with a desired target. With such probes, binding to the target either disrupts or initiates photophysical processes ,12,13, sIn an attempt to increase the sensitivity of turn-on fluorescent probes and to expand the mechanisms underlying their function, much attention has been devoted to the use of dyes governed by torsional motions as the candidates of choice for fluorescent sensing in biological environments ,21. ThisOwing to TO’s proven interactions with RNA and DNA, the detection of nucleic acids has long been the focus of TO-based sensors. This has brought about various sophisticated molecular devices that, in some cases, can detect even a single mismatched mutation. Nevertheless, TO fluorescence has also been shown to be induced by several other binding events that do not involve intercalation into nucleic acids, for example, interactions with supramolecular hosts or amino acid side chains. This has enabled the implementation of TO in sensing a variety of other types of analytes, such as ions, small molecules, and proteins. This review summarizes the use of TO for the sensing of biomolecules and ions, as well as highlights notable examples of its application in the field.Nucleic acids are undoubtedly the most thoroughly described targets of TO-based sensors. The dye was first proposed for the detection of reticulocytes using flow cytometry , replaciAlthough fluorescent probes for sensing nucleic acids have been in use long before the discovery of TO, these systems suffer from several limitations that can reduce their detection accuracy. For example, fluorophores such as DAPI, ethidium bromide, and the Hoechst family of dyes are commonly used for staining nucleic acids in gels or for imaging cell nuclei ,33. HoweA shared drawback of TO and other common dyes that are used in nucleic acid staining is their lack of structure or sequence specificity a. CurrenIn comparison with MBs, TO exhibits a very low initial fluorescence and a substantial increase in its emission upon binding to nucleic acids, which does not involve FRET. These properties have made the dye a leading candidate for designing advanced, specific, and low-background molecular probes able to sense DNA and RNA. Careful chemical modifications of TO have enabled the emergence of sensors that can be applied for in vitro detection of nucleic acids and even for imaging of RNA targets in living cells and tissues. These sensors can be classified into three main subtypes: (a) sequence-targeting probes, (b) structure-targeting probes, and (c) probes that can detect protein–DNA interactions. The first subtype relates to molecular hybridization probes directed at specific sequences of DNA or RNA, commonly employing complementary sequences for a desired target. The second subtype includes probes that can principally interact with certain nucleic acid structures and conformations, either in a statistically favorable manner or by using stabilizing agents. The last subtype employs TO-labeled DNA-binding proteins to sense the formation of protein–DNA complexes.Early attempts at designing TO-modified probes for sensing specific DNA sequences were carried out using peptide nucleic acid (PNA) chain-based hybridization modules, which were termed Light-Up probes a. IntrodDespite their success, Light-Up probes are generally limited by the relatively high background signal that they generate, which results from intramolecular interactions between the flexibly linked TO and the bases of the PNA probe . This, aDervan  and AsseIn an effort to prevent non-specific intramolecular activation of TO and to improve probe sensitivity, Seitz and his team established TO as a broad-use base surrogate that can be inserted within the probe’s sequence a. TO wasIt should be noted that the absorbance and quantum yield of TO in PNA-based FIT-probes can be affected by the neighboring base pairs in the formed complexes . GeneralK-ras mRNA in cancer cells. Both studies achieved impressive results with single-nucleotide resolution, enabling researchers to distinguish between cells expressing the wild-type sequence and cells expressing a SNP mutation. In another study, Yavin was able to use FIT-probes as a diagnostic tool for sensing the high expression of the long non-coding RNA (lncRNA) cancer-associated transcript 1 (CCAT1) . C. C65]. CDespite the various advantages of using PNA, the innate differences in the chemical properties of PNA-based probes from those of normal DNA and RNA may complicate detecting these targets in biological mixtures. As noted above, PNA-probes are more hydrophobic than DNA and RNA due to the lack of polar sugar-phosphate groups in their backbones. Consequently, this may lead to probes’ aggregation or cause some unspecific binding interactions. Thus, DNA-based TO-probes have been put forward as a means to more closely resemble natural nucleic acids. Compared to a PNA backbone, a DNA backbone is highly hydrophilic and hence, suitable for work in aqueous environments. On the other hand, its concentrated negative charge may lead to a high background signal owing to electrostatic interactions with the cationic TO dye. Moreover, a glycosidic bond between 2′-deoxyribose and a positively charged nitrogen of TO, as part of a surrogate base, would be very labile. As will be described hereafter, different strategies have been employed to solve such issues, yielding many types of DNA-based TO probes.oskar mRNA in a wash-free manner [Expanding their nucleic acid-sensing arsenal, Seitz developed a new set of FIT-probes based on DNA a. In orde manner . The same manner . The qFIO-propargylated uridine [Wagenknecht demonstrated an alternative 5-atom linear carbamate-bonded linker for attaching TO to DNA-probes ,71,72, a uridine  or an ar uridine  , from DNA conformation-modulating histone proteins that control the folding and accessibility of chromosomes and genes, to a myriad of replication and transcription machineries that control the output of DNA-coded instructions. To follow the functions of such proteins, different studies utilized TO-labeled DBPs in order to identify protein–DNA complexes . AlthougWoodbury conjugated TO to a peptidic zinc-finger binding domain of the glucocorticoid receptor . Upon biDespite the potential applicability of this approach, covalent attachment of TO onto a specific location in a protein is required, which poses several issues. Ensuring that the protein structure is not compromised by the labeling is crucial. Moreover, attachment of TO close to the DNA-binding domains, as is the case in the above examples, may induce promiscuity in DNA recognition of the probes, making them prone to reacting with random sequences, much like terminally TO-labeled DNA probes. It should also be taken into account, as explained in Understandably, TO has made its mark in DNA and RNA research, owing to its remarkable ‘turn-on’ fluorescent properties. Nevertheless, controlling TO’s fluorescence through intercalation is just one approach available to fine-tune the optical responses of TO-based fluorescent sensors. On its own, TO can be chemically modified to exhibit increased fluorescence in response to small molecules or ions. For example, a study of a TO-derivative appended by two 17-atom-long aliphatic chains found that it exhibited selective increased fluorescence in the presence of ATP, relative to AMP, ADP, and other nucleoside triphosphates .Another approach to sense small molecules concerns the dimerization and aggregation properties of TO, a phenomenon termed aggregation-induced emission (AIE) . This apHeyne  showed t+ and Ca2+ ions at high CB7 concentrations: Na+ ions yielded turbid particles, while Ca2+ ions retained solubility with increased H-dimer fluorescence. This observation was attributed to different ion-mediated assemblies being stabilized by the two ions, positioning TO host-guest probes as potential analytical tools for detecting metal ions.Subjecting the 2CB8:2TO complexes to biologically relevant analytes, such as tryptophan or sodium ions, resulted in decreased fluorescence and distinct absorption shifts, indicating that different analytes are identified via distinct TO-release mechanisms a, right.TO’s outstanding interactions with DNA and RNA were also harnessed to detect ions and small molecules. Stojanovic  prepared2+ in solution  revolutionized biology by enabling the monitoring of protein expression and dynamics, both at spatial and temporal resolutions . FPs, suIn an effort to image specific proteins in living cells while refraining from their fusion to large protein tags , an alternative tagging method emerged. With this approach the target proteins are expressed with an added short amino acid sequence-tag that can be selectively labeled by an externally added fluorescent probe ,144, ford value of 1.7 nM and induces a 2600-fold increase in TO’s fluorescence.Naturally, the low background emission of TO and its strong ‘turn-on’ fluorescence response make it an ideal candidate for labeling fusion tags in living cells. This was achieved by Waggoner , whose tBy expressing a FAP-modified platelet-derived growth factor receptor (PDGFR) in living cells and subjecting the cells to the TO-based fluorogen, the receptor’s membrane localization and its secretory pathway were visualized. As the fluorescence of FAPs depends on the binding of externally added TO, FAPs are more resistant to photobleaching effects, since photobleached molecules can be conveniently replaced by free TO in the solution. However, the reported TO-binding FAP is similar in size to GFP, maintaining the need to find fluorophore-activating modules that are at least as bright yet smaller. Overall, FAPs have been shown to be equally as effective as traditional fluorescent proteins in the labeling of cellular protein targets, with the added features of tunable and reversible fluorophore binding. In a subsequent work , TO-bindUnlike FPs, which possess autonomous fluorescence, nucleic acids lack such fluorescent counterparts. Consequently, the detection and imaging of DNA and RNA inside living cells has proven to be challenging. In the quest to develop suitable fluorescent probes for these biomolecules, several methods have been described and thoroughly reviewed ,157,158,In an attempt to fill this void, GFP has been utilized as part of a well-implemented technique for sensing RNA molecules in cells, by fusing it to a bacteriophage-derived MS2 protein ,160. MS2In the RNA-of-interest, the stem-loop that binds MS2-GFP is essentially a RNA aptamer that can specifically bind the MS2 protein. Therefore, an interest arose in using small fluorescent dyes, which light up upon binding to specific RNA aptamers, as a possible replacement of the GFP fusion protein . Many suFollowing these studies, novel RNA aptamers, termed Mango RNAs, which can tightly bind and activate TO, were developed ,170,171 Examples mentioned above of protein labeling , using TTo overcome these limitations, our group has developed TO-based sensors that can detect native (non-engineered) proteins. Specifically, we have shown that modifying TO with specific protein binders affords a new class of ‘turn-on’ probes, termed TO-based protein identifiers TOPIs, a, which Perhaps most strikingly, certain TOPI probes displayed an isoform-selective fluorescent response b. For exThis review summarizes research involving the use of TO as an essential component for creating ‘turn-on’ fluorescent probes for the sensing of a wide range of biomolecules and ions. Compared to traditional fluorescent dyes, the torsional control over the fluorescence of TO has the advantage of being internally embedded within the molecular structure of the dye. Therefore, TO-based fluorescent sensors do not necessarily require secondary auxiliary units to control their signal, in contrast with existing FRET-based probes. Additionally, the very low emission of TO in aqueous environments and the large increase in the dye’s fluorescence in response to restrictive interactions, sometimes reaching over 1000-fold, provides remarkable signal sensitivity through high S/N ratios. Finally, TO offers great versatility through chemical modifications that can be utilized to adjust its fluorescence response to different types of biomolecular targets.The most detailed use of TO lies in the sensing of nucleic acids, for which TO’s intercalating nature has been well studied and understood. This has enabled TO to evolve from a simple DNA staining dye into a powerful tool that can be applied in several biochemical research methods, including those involving probes that have impressive sensitivity of up to a single nucleotide mismatch. However, the expanding application of TO to small-molecule and protein detection, as well as to sense specific nucleic acid conformations, leaves many unanswered questions regarding these less familiar interactions. Revealing the nature of these interactions is expected to lead to the development of even more accurate, sensitive, and selective TO-based probes.In conclusion, we hope that this review will serve to publicize the advances made so far, and that it will inspire future endeavors that will contribute to our growing understanding of the mechanisms underlying TO-based probes and the ways to apply them in various areas of analytical biosciences.