Laser speckle contrast imaging (LSCI) is a powerful tool to monitor blood flow distribution and has been widely used in studies of microcirculation, both for animal and clinical applications. Conventionally, LSCI usually works on reflective-detected mode. However, it could provide promising temporal and spatial resolution for in vivo applications only with the assistance of various tissue windows, otherwise, the overlarge superficial static speckle would extremely limit its contrast and resolution. Here, we systematically investigated the capability of transmissive-detected LSCI (TR-LSCI) for blood flow monitoring in thick tissue. Using Monte Carlo simulation, we theoretically compared the performance of transmissive and reflective detection. It was found that the reflective-detected mode was better when the target layer was at the very surface, but the imaging quality would rapidly decrease with imaging depth, while the transmissive-detected mode could obtain a much stronger signal-to-background ratio (SBR) for thick tissue. We further proved by tissue phantom, animal, and human experiments that in a certain thickness of tissue, TR-LSCI showed remarkably better performance for thick-tissue imaging, and the imaging quality would be further improved if the use of longer wavelengths of near-infrared light. Therefore, both theoretical and experimental results demonstrate that TR-LSCI is capable of obtaining thick-tissue blood flow information and holds great potential in the field of microcirculation research. The performance of novel transmissive-detected LSCI was systematically demonstrated through simulation and experiments. With such a simple system, individual vessel-resolution blood flow mapping and monitoring were realized on human hand. Conventionally, it works on the reflective-detected mode, and has been widely used in the fundamental research of microcirculation7, whose dysfunction is highly relevant to a series of clinical symptoms, such as diabetes8, ischemic stroke10, coronary heart disease11 and peripheral artery disease12. With surgery-based open-skull windows, thinned-skull windows, and surgery-free skull optical clearing windows, cortical blood flow distribution could be clearly observed using conventional reflective-detected LSCI technique13. With skinfold chamber windows and skin optical clearing windows, conventional LSCI could also provide cutaneous blood flow mapping with individual-blood-vessel resolution14. However, without such “windows”, the light should penetrate the upper tissue layer above the deep blood vessel layer, during which path it constantly decays, making the strength of static speckle in the upper layer much greater than that of dynamic speckle signal in the deep targeted layer, leading to the extremely decreased contrast and resolution of conventional LSCI, or even making the blood flow undetectable15. Moreover, even with the assistance of skull and skin windows, conventional LSCI is still only able to provide acceptable resolution in the superficial layers, while even the body parts of mice are often hundreds of microns or even millimeters thick, making it barely possible to obtain comprehensive information using such a technique18.Laser speckle contrast imaging (LSCI) is a wide-field, noninvasive imaging technique with high temporal and spatial resolution, which is based on the analysis of light signals after scattering and random interference, and therefore obtains the velocity information of scattering particles in biological tissues 19, while the imaging quality improvement was still limited. Later, there were preliminary results which indicated that changing the direction of incident light might contribute to the extraction of thick-tissue information21. It was observed that transmissive-detected laser speckle contrast imaging (TR-LSCI) showed better imaging contrast and resolution than conventional LSCI on blood flow mapping in the hindlimb of mice and could even provide microvascular functional imaging in human finger joints without other supplementary means. However, such discoveries have not got many attentions, and the TR-LSCI technique has not been thoroughly studied, therefore the mechanisms why TR-LSCI might be promising for blood flow monitoring in thick tissue remain unclear. What is the effect of the depth and thickness of the target tissue on these two imaging modes? What are the factors those could influence the performance of TR-LSCI? Is there a situation where the imaging quality of conventional LSCI is better than that of TR-LSCI? The answers to these questions will enrich the theory of laser speckle imaging for biological tissues.In order to overcome such a problem, researchers further looked to algorithms for solutions, such as calculating temporal contrast instead of spatial contrast analysis to reduce the effect of static speckle22, a powerful tool to simulate the optical propagation and distribution in the tissue, was used to reveal the law of the signal-to-background-ratio (SBR) of reflective- and transmissive-detected imaging modes with varying signal depth and tissue thickness, mechanically illustrating why TR-LSCI is more suitable for thick-tissue imaging. Next, a series of experiments on 3 levels, including tissue phantom level, animal level, and human level, were designed to evaluate the validity of the Monte Carlo simulation results. The two imaging modes were firstly compared in the intralipid with the change of thickness and signal depth. Secondly, cutaneous/subcutaneous blood flow mapping in different mice body parts  was performed. Finally, conventional LSCI and TR-LSCI were applied in human hand imaging (finger and opisthenar). All the experimental results well fit the conclusion of the Monte Carlo simulation. Therefore, this work compared the performance of conventional LSCI and TR-LSCI when used in bioimaging and provides a guidance for researchers to choose a proper imaging technique to achieve better imaging quality in thick tissue, which is significant in in vivo microcirculation studies.This work aims to systematically evaluate the capability of TR-LSCI for thick-tissue observation from theory to practice. Here, Monte Carlo techniqueFigure 23 we also performed comparison between conventional LSCI and TR-LSCI with a polarizing filter matching the light source in front of the detector. The results indicated that, the polarizing filter could indeed improve the performance of both two modes. In addition, the comparison between two imaging modes was similar to that without the polarizing, where TR-LSCI was comparable at surface, and performed much better at large depth. , and the blood flow velocity was set from 0 to 4 mm s24.Here, the speckle sizes of conventional LSCI and TR-LSCI were calculated to be similar in our experimental condition (2.50 pixels vs. 2.45 pixels with ×2 magnification). Since it has been demonstrated that the speckle size would influence the sensitivity of conventional LSCI to the change of velocity, it is valuable to explore the influence of speckle size on TR-LSCI. As shown in Fig. S25, hindlimb26, dorsal skin27, and paw7 of mice  was presented in Fig. −2 vs. 6 mW cm−2). In order to exclude the influence of power density on conventional LSCI, we further improved its power density to the same as TR-LSCI, and also found it could not distinguish vessels in the human finger  which was much lower than that used in low-level-laser therapy 29.Furthermore, in order to evaluate the safety of TR-LSCI on human imaging, we investigated the proper light power density as well as exposure time for TR-LSCI on human hand. As shown in Fig. Next, the ability of TR-LSCI to monitor vascular functional response in human hand was tested. Reactive hyperemia experiments were performed and the results were shown in Fig. Other than cuffing experiments, we were also able to monitor blood flow velocity changes of human hand after exercise using TR-LSCI. As shown in Fig. SSuch results suggested that TR-LSCI provided a powerful tool for monitoring hemodynamic change in human hands.20 observed that changing the position of the detector from the same side to the opposite side of the light source could improve the performance of LSCI and applied it to the acquirement of vascular functional information in human digital joints. Meisner et al.21 found TR-LSCI could be used to visualize blood flow in the mice hindlimb. However, neither of them has systematically explored the difference in the performance between TR-LSCI and conventional LSCI. Herein, we systematically demonstrated the potential of TR-LSCI in thick-tissue blood flow mapping. Using simulation, we clearly revealed that the ratio of the light signal from the target layer to the light signal from the whole tissue might be a significant factor that decided the imaging quality. Since the light attenuated quickly, for reflective mode, when penetrating a highly scattered tissue from the surface, the light reaching the superficial layer was much stronger and the secondary attenuation before received by the detector was much smaller, thus the imaging quality of the superficial layer could be desirable, while it would rapidly decrease when the interested layer goes deeper. On the contrary, for transmissive mode, although the secondary attenuation of the signal from the deeper layer before detected was stronger, the light irradiation was also stronger than that of the shallow layer, making the imaging quality for deep tissue also promising. Such results of simulation indicated that conventional LSCI would be superior when the ROI is in the very surface of tissue, but had shortcomings for deep signal without any further treatment to the upper layer (e.g. removing the upper tissue or make it transparent8), while TR-LSCI holds advantages in deep-tissue monitoring for a certain thickness of tissue.We systematically evaluated the capability of TR-LSCI for thick-tissue blood flow monitoring, from Monte Carlo simulation to experimental demonstration. Dunn et al.31 which might suggest that TR-LSCI could hold advantage over conventional LSCI for deep detection because TR-LSCI mainly uses forward-scattered light while conventional LSCI uses backscattered light. Our study and other studies have shown that a polarizing filter would improve the performance of LSCI, which also provides support for this conjecture. In addition, further simulations and experiments should be performed to mechanically discuss the dynamic light scattering behind the TR-LSCI, like how many scattering events are expected for thick tissue, and what the correlation functions at different depths should be, similar to what was done for conventional LSCI.32 In this case, TR-LSCI can be further studied and optimized.Other than the difference between two modes on the ratio of deep signal, there might be other factors those impact the performance of conventional LSCI and TR-LSCI. For instance, LSCI is a technique that highly relies on the interference of light. However, light will become less polarized experiencing scattering events in tissue, which will be adverse to interference and so as to LSCI. It has been found through Monte Carlo simulation that compared to backward scattering, forward scattering suffers a lot much less depolarization when experiencing the same number of scattering events,21. Here, mice body parts with various thicknesses, including the ear, hindlimb, back and paw were chosen as imaging sites to evaluate the validity of our simulation, as well as the capability of TR-LSCI for monitoring the targets where structural and functional information of blood vessels is always desired by researchers in the basic research of microcirculation27. In addition, different positions of human hand were also investigated. Our systematical in vivo results also suggested that TR-LSCI could perform blood flow mapping with promising resolution in thick tissue without the assistance of tissue windows, those conventional LSCI needs for the research of tumor vasulature34, wound healing35, therapeutic effect of thrombolytic drugs37, photodynamic therapy of malformed blood vessels40 and so on.For the experimental study of TR-LSCI, the previous works were not systematically designed, but just chose one part of mice or human body41, rheumatoid arthritis42, and dermatitis43. Moreover, our results showed that, 850-nm wavelength LD as imaging light provided better imaging quality than that of 785 nm, which we attributed to the greater penetration depth at 850 nm. In fact, NIR-II (900–1700 nm) light has even more promising penetration and has been widely used in deep-tissue imaging in recent years44. Compared to NIR-IIb (1560–1700 nm), where the high absorption of tissue might limit the application of TR-LSCI, NIR-IIa (900–1300), especially 1300 nm, has relatively low absorption and acceptable scattering in tissue. Consequently, TR-LSCI using such wavelength range might hold great potential for high-resolution blood flow mapping in further clinical use. Nevertheless, it should be noticed that a promising detector is important for human TR-LSCI imaging. Herein, using a high-end CCD that responded more than 90% in 400–900 nm wavelength range, the imaging laser power density could be set as low as 30–60 mW cm−2. However, it might not directly work for cameras typically used in conventional LSCI, especially in the NIR region, where it would require higher light power density for human applications.Furthermore, the successful acquirement of blood flow signal in human hand implied that TR-LSCI might be further applied in other human body parts whose thickness is feasible for light to penetrate, such as ear, lip, toe, and instep etc. Thus, the development of TR-LSCI might accelerate the clinical research of microcirculation and related diseases, such as diabetic foot ulcerAdmittedly, TR-LSCI has its own limitations. Like conventional LSCI, TR-LSCI cannot distinguish the depth of vessels, nor allow to measure absolute blood velocity, but it is only capable of qualitative analysis of blood flow dynamics. Furthermore, although our study clearly demonstrated that, TR-LSCI could obtain high-resolution blood flow information in tissue with a certain thickness and optical properties, such as the ear, hindlimb, paw, back of mice, and human hand or perhaps other small human body parts in future, when the tissue thickness or the scattering is overlarge, it might be better to use conventional LSCI with a tissue window. For instance, for mice brain monitoring, as shown in Fig. SIn summary, our study revealed an alternative of LSCI for noninvasive thick-tissue blood flow imaging and discussed its proper applications with simulations and experiments. As thus, according to the specific application, a more suitable imaging scheme can be selected between conventional LSCI and TR-LSCI.45, the refractive index, absorption coefficient, scattering coefficient, and anisotropy factors were set as Table Here, Monte Carlo simulation was used to theoretically compare reflective and transmissive imaging modes in thick tissue. According to the optical characters of skin to 633-nm lightThe light source was set as an infinitely narrow photon beam, which was incident perpendicular to the tissue. Two series of simulations were performed. The first one was based on a single-component tissue , and the second one was based on a multi-component tissue: stratum corneum (20 μm)-epidermis (80 μm)-dermis (520 μm). To simplify the model, we discussed only seven layers in the stimulated single-component tissue, those were located at 0, 100, 200, 300, 400, 500, and 520 μm, respectively, and discussed nine layers in the stimulated multiple-component tissue, those were located at 0 (the surface of stratum corneum), 20 (the surface of epidermis), 100 (the surface of dermis), 200, 300, 400, 500, 600, 620 μm, respectively.For reflective imaging, we assumed that the light was irradiated from the top of the tissue, and the desired signal from the target layer, as well as the background signal from other layers, was detected at the top of the tissue. To calculate the ratio of the signal from the target layer to the signals from all the seven layers , a two-step Monte Carlo simulation was performed. Firstly, we simulated the luminous flux distribution of light as it penetrated the entire tissue, thereby obtaining the luminous flux ratio of the seven layers described above. Secondly, we set the initial light flux of each layer according to this ratio to simulate the remaining light flux when the light of each layer returning to the surface. In this case, we could obtain the ratio of the luminous flux returned from every layer, and the SBR could be calculated when any layer was set as the target layer.The simulation and calculation of the SBR of transmissive imaging were similar, where the differences were that the light was irradiated from the bottom of the tissue, and the desired signal from the target layer as well as the background signal from other layers was detected at the top of the tissue.To investigate the effect of the thickness above or below the target layer on the SBR of reflective- and transmissive-detected modes, we chose the target layer at 200 μm in the 520-μm-tissue as the initial situation where their SBR was approximate. Then the position of the signal layer kept constant and the thickness above or below the signal layer increased to 400 μm taking 100 μm as the step size. The simulated tissue parameters remained the same as previously mentioned and the SBR of two imaging modes when the tissue had different thicknesses was calculated, respectively.To perform the Mento Carlo simulation corresponding to the tissue phantom experiment, we first constructed the simulated tissue of 5 mm and divided it into 11 layers those were located in 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mm, respectively, to simulate the situation where the total thickness was constant, but the signals depth varied from 0 mm to 2.5 mm. Then we constructed another simulated tissue to simulate the situation where the position of the signal was constant and the thickness of the upper layer of the signal continuously increased. The tissue was first set as 3.5-mm thick and simplified to 8 layers located in 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 mm. The target layer was fixed at 0 mm of such tissue, and then the upper thickness of the tissue increased by 0.5 mm every time until the whole tissue thickness was 5.5 mm.13:Kt was the temporal contrast at pixel , σx,y) was used to simulate the blood vessel embedded in the turbid tissue. The capillary glass tube was insert diagonally into a culture dish . Blood was taken from the caudal venous plexus of mice and mixed with heparin sodium  to prevent clotting. After drawing mice blood, a micro syringe was clamped on the micro syringe pump, and its needle tip was inserted into the capillary glass tube. Then, the blood was pumped into the capillary with certain velocities.As shown in Fig. Another kind of tissue phantom was constructed by inserting the capillary glass tube into the culture dishes horizontally. The bottom of the capillary was 2.5 mm from the bottom of the dish and the thickness  of the mixture of intralipid and agarose was changed by adding a mixture of different volumes to culture dishes. The capillary glass tubes in these two kinds of tissue phantom were imaged with TR-LSCI and conventional LSCI equipped with the 785-nm LD and blood flow maps were acquired. Then, we evaluated the imaging quality by subtracting background from the signal.All animal procedures were approved by the Experimental Animal Management Ordinance of Hubei Province, China, and carried out in accordance with the guidelines for humane care of animals. Mice  were anesthetized by gas anesthesia machine (1.5% isoflurane) during experiments. The hair on ear, hindlimb, back and paw was shaved and the remaining hair was cleaned by treating with depilating cream. Mice ear, hindlimb and paw were placed directly on the experiment platform, respectively. In specific, to perform blood flow imaging in dorsal skin, two-fold skin at the shaved spot was lifted and fixed with an aluminum bracket. The raw speckle images were acquired with TR-LSCI and conventional LSCI under the irradiation of 785-nm laser.−1) and a continuous recording for 11 min. We acquired the blood flow intensity in the blood flow maps at every time point with MATLAB and calculated the relative changes of blood flow (Δ Flow), which was defined as:Ii was the blood flow intensity at a certain time point and IBL was the baseline value, that is, the average value of blood flow intensity before stimulation.To test the capability of LSCI to dynamically monitor the response of blood flow to drugs in mice, the blood flow of the subcutaneous blood vessels in mice hindlimb was recorded in its resting state for 160 s, followed by a vein injection of 125 μL of Ach  and then released, during which period the blood flow was monitored while the pressure was recorded. For squat experiment, after acquiring blood flow imaging in the resting state, the volunteers did squats for 2 min, and then we monitored the change of blood flow in the same place during the next 5 min. The calculation of relative changes of superficial perfusion and blood flow was similar to that of animal experiments.Supplementary information


Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that have been associated not only with various cellular processes, such as embryonic development and adult wound healing but also enhanced tumor survival, angiogenesis, and metastatic spread. Proteolytic cleavage of these single-pass transmembrane receptors has been suggested to regulate biological activities of their ligands during growth and development, yet little is known about the proteases responsible for this process. In this study, we monitored the release of membrane-anchored FGFRs 1, 2, 3, and 4 in cell-based assays. We demonstrate here that metalloprotease-dependent metalloprotease family, ADAM10 and ADAM17. Loss- and gain-of-function studies in murine embryonic fibroblasts showed that constitutive shedding as well as phorbol-ester-induced processing of FGFRs 1, 3, and 4 is mediated by ADAM17. In contrast, treatment with the calcium ionophore ionomycin stimulated ADAM10-mediated FGFR2 shedding. Cell migration assays with keratinocytes in the presence or absence of soluble FGFRs suggest that ectodomain shedding can modulate the function of ligand-induced FGFR signaling during cell movement. Our data identify ADAM10 and ADAM17 as differentially regulated FGFR membrane sheddases and may therefore provide new insight into the regulation of FGFR functions. Fibroblast growth factor receptors, a family of high-affinity cell surface receptor tyrosine kinases (RTKs), are essential regulators of critical cellular processes such as cell proliferation and differentiation and have important roles in embryonic development and cancer ,2,3. TheEctodomain shedding of RTKs has been shown to be a posttranslational modification to rapidly and irreversibly regulating cell surface expression levels ,7,8. ThiEctodomain shedding is mainly executed by the a disintegrin and metalloprotease (ADAM) family of metalloproteases. ADAMs are members of the zinc protease superfamily and combine both cell adhesive and catalytic properties. They perform important functions including fertilization, angiogenesis, and wound healing ,16. CataThe main goal of the current study was to evaluate what role, if any, ADAMs have in the ectodomain shedding of FGFR family members. We demonstrate that metalloprotease-dependent cleavage of FGFRs 1–4 is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. Loss- and gain-of-function studies in murine embryonic fibroblasts showed that constitutive shedding as well as phorbol-ester-induced processing of FGFRs 1, 3, and 4 is mediated by ADAM17. In contrast, treatment with the calcium ionophore ionomycin stimulated ADAM10-mediated FGFR2 shedding. This shedding process modulates FGF7/FGFR2-mediated cell migration and suggests that soluble forms of FGFRs can act as antagonists, preventing interaction of the membrane-bound forms of FGFRs with their ligands.Previous studies have shown that the extracellular domains of all four FGFR family members can be released through membrane-proximal cleavage, leading to generation of C-terminal fragments and soluble N-terminal fragments (NTFs) ,16,30. TIn-gel AP staining of supernatants of AP-FGFR-expressing COS-7 cells demonstrated that the release of FGFR1c A, 2c FiB, 3c FiC, and 4-This observation, especially when combined with the fact that other RTK substrates are cleaved by similar proteases , suggestADAM10−/− and ADAMs 9/15−/− mEFs and comparable with WT mEFs. In contrast, ADAM17−/− cells showed significantly reduced generation of soluble N-terminal fragments (−/−ADAM10 cells (ADAMs 9/15−/− or ADAM17−/− mEFs (ADAM17−/− cells was indeed due to lack of ADAM17. Transient transfection of WT ADAM17 (WT A17) rescued constitutive shedding in ADAM17−/− mEFs. Similarly, we found that the defect in FGFR2c shedding in ADAM10−/− cells was due to lack of ADAM10  deficient for either ADAM10, ADAMs 9/15, or ADAM17 with WT cells. Constitutive shedding of FGFRs 1c A, 3c FiC, and 4 ragments A,C,D. Mo/− cells B, but no−/− mEFs B. To exc−/− mEFs E, 3c  using phorbol ester phorbol-12 myristate 13-acetate (PMA) as well as ionomycin (IO), an agent that promotes shedding through stimulation of calcium influx , stronglADAM17−/− mEFs  or the calcium ionophore IO (ADAMs 10 and 17) within 30 min ,34. Our inactive . Our finWhile our study focused on the proteolytic processing of the FGFR c splice isoforms, in future studies, it will be important to determine if the b splice isoforms of these receptors are shed by the same proteases in a similar manner. In common with our current findings, we have previously shown that the b splice isoform of the FGFR2 is also shed by ADAM10 , suggestPrevious studies have shown that FGFRs can be shed from the plasma membrane and are found in a secreted, soluble form , yet litADAM10- and ADAM17-deficient mice will further aid in dissecting the role of these metalloproteases in regulating FGFR-mediated cell growth and division in vivo.While the physiological relevance of ADAMs 10 and 17 in processing FGFRs remains to be determined, it is tempting to speculate that these metalloproteases could contribute to the downregulation of the membrane-anchored form of the receptor, at least in cells or tissues in which both proteins are highly expressed. Additional studies in conditional ADAM9/15−/− E13.5 embryos and cultured as described previously [ADAM9/15−/− mEFs, we also used ADAM17−/− mEFs [ADAM10−/− fibroblast cell lines derived from E9.5 embryos have been described previously [Simian virus large-T-antigen-immortalized mouse embryonic fibroblasts (mEFs) were generated from wild type (WT) or eviously ,54. In a−/− mEFs ,55 as weeviously . COS-7 ceviously ,44.XhoI and/or XbaI sites of pKitl2APtag5 vector [The expression vectors for ADAMs 9, 10, 12, 15, and 17 have been validated for cell surface localization and proteolytic activity previously ,35,56,575 vector  in frame5 vector . In bothMouse embryonic fibroblasts or COS-7 cells were transiently transfected with Lipofectamine 2000 , per manufacturer’s protocol, using ADAM WT or catalytically inactive (EA) plasmids at a concentration of 1.5 μg per well of a 12-well plate, unless otherwise indicated, and FGFRs 1 to 4-AP vectors at 1 μg. Shedding assays were performed the day after transfection ,35,53,59In-gel detection of alkaline phosphatase-labeled membrane proteins was performed as previously described ,60,61. Bhttps://imagej.nih.gov/, accessed on 17 December 2020) was used for quantification of scratch wound assays [Cells used for in vitro scratch wound-healing assays were plated in 12-well plates and grown until they reached confluence. A thin scratch wound was introduced by scratching with a 200 μL pipette tip. After washing with phosphate-buffered saline, cells were incubated with or without FGF7, soluble forms of FGFRs 2 and 3, or IO as indicated. Images were taken using a digital transmitted light, inverted imaging system , and NIH Image J software  following manufacturer’s protocol. RNA quantity and quality were measured with a NanoDrop ND-1000 spectrophotometer . Pre-verified qPCR primers for FGFR1–4 and ADAMs 9, 10, 12, 15, and 17 were purchased from MilliporeSigma  . Gene transcripts were measured in triplicates on a real-time PCR detection system  using a PerfeCTa SYBR® Green FastMix  and normalized to the reference gene Beta-actin. Thermocycling conditions were set as follows: 1 cycle (95 °C for 5 min), 40 cycles .Total RNA from HaCaT cells was extracted using the RNeasy Plus Mini kit , and cDNA was produced with 500 ng RNA using a reverse transcription kit . All values are expressed as means ± standard error of the mean (SEM). One or two-way analysis of variance (ANOVA) testing was performed. Multiple parametric statistical comparisons between experimental groups versus a control group were accomplished by Dunnett’s method. All pairwise multiple comparison procedures were performed via Bonferroni’s multiple comparison test. Statistics following a Student’s In summary, this study demonstrates that membrane-anchored members of the FGFR family are shed from various cell types, including COS-7 cells and mEFs, and identifies the responsible sheddases. The release of soluble forms of FGFR should decrease their availability on cells and simultaneously generate soluble decoys that could regulate the biological activity of members of the FGF family of growth factors, similar to other soluble RTKs.


The escape of a particle from a dynamical system depends on the intersection between the ingoing and outgoing asymptotic trajectories to certain periodic orbits placed at the openings of the curves of zero velocity of the system. Although many efforts have been devoted to the analysis of the escape from potentials presenting multiple openings, there are still few studies on potentials with only one opening. In this article, we clarify the way in which the energy affects the escape in this type of systems, showing that, contrary to what one could expect, there are several bifurcations for certain values of the energy. In these systems, the motion of the particle is limited by the curves of zero velocity. These curves, for certain values of the energy of the system, open creating windows, and particles can leave the system through them. There is one unstable periodic orbit, called Lyapunov orbit, located at any of the openings of the curves of zero velocity of the system. If the test particle crosses one of these orbits, then the particle leaves the potential well. In particular, the escape of a particle from a system depends on the relation of its initial conditions with respect to the stable manifolds to the Lyapunov orbits. If the initial conditions belong to the inner part of these structures, then the orbit will escape and, if not, it will remain trapped forever.The motion of a particle in a gravitational potential and its possibility of escape from it has been extensively studiedMost of the works devoted to escapes from dynamical systems analyze systems that present multiple openings. In these systems, the way in which the escape occurs depends on the energy of the system, since a variation in its value makes the intersections of the asymptotic curves of the different Lyapunov orbits vary considerably. However, few works have been dedicated to the study of systems with only one exit channel. We think that this type of potentials have the property of showing in a simple way the logic that explains the escape of a particle from the system. It remains to be studied if the variation of the energy produces a variation in the form in which the escape takes place in this type of systems. The object of this paper is to clarify this question.19 and Navarro13. In a companion paper, Navarro13 investigates the geometry of the curves that delimite the escape domains by determining the intersection of the ingoing and outgoing asymptotic trajectories to the Lyapunov orbit with an apropiate surface of section, for a fixed value of the energy. In this paper, we describe how these limiting curves evolve as the energy of the system varies, showing that the intersection between the ingoing and outgoing asymptotic trajectories to the Lyapunov orbit takes place in a way that relies on the energy of the system.To this end, we study the escape from a galactic model with axial symmetry. This type of galactic system has been previously studied by Zotosr, z) meridian plane near the central part of an axially symmetric galaxy modeled by a galactic type potential of the formr and z axis, where r and z are cylindrical coordinates, 19. The potential  is axially symmetric and the 19H is the numerical value of the energy of the system, which is conserved. We can obtain the curves of zero velocity by substituting H, there is an unstable periodic orbit at the exit of the potential.As entclass1pt{minima13. In Fig. In our work, we use a system of galactic units, where the unit of length is 1 kpc, the unit of time is 21. The selection of the initial conditions of these curves have been determined by following the procedure described by Navarro13. We have computed and integrated backward a set of As we have stated above, the asymptotic trajectories to the Lyapunov orbit constitute the limit of the set of escaping orbits. It is common to use an adequate surface of section in order to unveil the geometry of these complex sets by analyzing their successive intersections on it. These crossings between the stable manifold to the Lyapunov orbit and the surface of section define the contour of the basins of escape, that is, the set of initial conditions leading to escape. For the computation of the initial part of these asymptotic curves, we follow the method suggested by Deprit and HenrardIn the following, 13. Orbits with initial conditions belonging to the area enclosed by The sets entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaH considered, Thus, we can integrate backward the two tongues colored in blue in Fig. entclass1pt{minima13. However, we can clearly observe that for a range of values of the energy in a neighborhood of Contrary to what happens with } \in -0.,0.5$$\enOrbits starting in the area delimited by H near Next, we will show how the hiding of the “tip” of the infinite tongues that make up In Fig. H in a neighborhood of H slightly larger than We observe this same phenomenon for values of If If Therefore, depending on whether Thus, we can conclude that we can find two different scenarios, both depicted in Fig. Finally, we observe that when we increase the energy of the system, the amount of orbits that escape varies substantially. To show this, we have calculated the percentage of orbits that escape directly without intersecting the surface of section, by considering a mesh of In the right panel of Fig.  The study of escapes in dynamical systems presenting one sole exit channel is a topic that has not received much attention by the scientific community. However, we think that these systems can exhibit very clearly the logic that explains the escape of a particle from the system. In this paper, we analyze this question, clarifying if the variation of the energy produces a variation in the way in which the escape takes place in this type of systems. To this end, we have studied the escape from a galactic model with axial symmetry, by investigating the geometry of the limiting curves of the escape domains by determining the intersection of the ingoing and outgoing asymptotic trajectories to the Lyapunov orbit with the surface of section. We have shown that, in fact, there are two intervals of values of the energy where the intersection between the ingoing and the outgoing asymptotic trajectories to the Lyapunov orbit takes place in a different way.


Rheumatoid arthritis (RA) is a chronic autoimmune disease that is primarily characterized by synovial inflammation. Our previous studies demonstrated that the lymphatic system is critical for the development and maintenance of RA disease, and sufficient lymph drainage helps to improve joint inflammation. In this study, we found that NG-R1, the main active component in the traditional Chinese medicinal herb Sanchi, activating lymphatic function can attenuate synovial inflammation. According to histopathological staining of ankle sections, NG-R1 significantly decreased the area of inflammation and reduced bone destruction of ankle joints in TNF-Tg mice. Near infrared-indocyanine green (NIR-ICG) lymphatic imaging system has shown that NG-R1 significantly improved the lymphatic drainage function. However, the molecular mechanism of its activity is not properly understood. Our in-depth study demonstrates that NG-R1 reduced the inflammatory cytokine production of lymphatic endothelial cells (LECs) stimulated by TNF-α, and the mechanism ameliorated the phosphorylation of IKKα/β and p65, and the translocation of p65 into the nucleus. In summary, this study proved that NG-R1 promoted lymphatic drainage function to ameliorating rheumatoid arthritis in TNF-Tg mice by suppressing NF-κB signaling pathway. Rheumatoid arthritis is a chronic systemic disease with autoimmune pathogenesis and systemic involvement, which is associated with the overexpression profile of many proinflammatory cytokines . This auThe lymphatic system plays a key role in the pathogenesis and treatment of inflammatory and invasive arthritis, and it has been proved that improving lymphatic drainage function effectively reduced RA joint inflammation , 541–553Panax notoginsen (Burk) F. H. Chen (Sanqi in Chinese) is a traditional Chinese medical drug which has the effect of promoting blood circulation and removing blood stasis  mice were provided by the Shanghai Southern Model Animal Laboratory Center . The TNF-Tg mice were bred as heterozygotes on a C57BL/6 background, and their wild-type (WT) littermates were used as normal controls. Mice were housed in a temperature (25°C) and humidity (45–55%)-controlled environment and provided libitum access to maintenance diet and water. All animal procedures were performed according to the Guiding Principles for the Care and Use of Laboratory Animals approved by the Animal Regulations of the National Science and Technology Committee of China under the approval of the Longhua Hospital Animal Ethics Committee .TNF-Tg mice were randomly divided into two groups: TNF-Tg group and NG-R1 group (10 mice/group). A batch of littermates of wild type (WT) mice were used as the WT group (10 mice/group). The TNF-Tg group and WT group were treated with the same volume of 0.5% CMC-Na (0.5 g CMC-Na + 99.5 ml aqueous solution + 500 µl tween). NG-R1 group mice were treated with 20 mg/kg NG-R1 (2 mg NG-R1 + 1 ml 0.5% CMC-Na) by daily intraperitoneal injection and continuously for 8 weeks.We conducted the measurements of arthritis index, ankle circumference, and weight. Arthritis disease malformation grade assessment: arthritis grade 0, normal ; grade 1, mild, but marked redness of the ankle joint or wrist swelling, or significant redness and swelling or malformation limited to individual figures, regardless of the number of affected numbers; grade 2, moderate redness and swelling or malformation of the ankle or wrist; grade 3, severe redness and swelling or malformation of the entire paw, including number; grade 4, the limbs are most inflamed and multiple joints. Body weight: an electronic body weight scale will measure weight once a week. Ankle circumference: the degree of ankle joint swelling was measured with a vernier caliper. The counts and grades recorded by two independent blinded examiners once a week.Mice were anesthetised with isoflurane and treated with local depilation, then ICG  was injected into footpads. After the ICG signal was stable, the fluorescence signal was observed for 600 s continuously, and the pulse number was obtained according to the fluctuation times of the signal intensity of lymphatic vessels. The ImageJ software  was used to determine the region of interest (ROI) during the lymph flow in the leg of mice, and the fluorescence signal intensity within the ROI was recorded at 1 and 24 h. Lymphatic clearance was calculated according to the following formula: clearance = ((ROI1h−ROI1hbg)-(ROI24h− ROI24hbg))/(ROI1 h − ROI1 hbg)*100% (bg = background).After ending treatments, the mice were sacrificed, and the ankle joints were excised to prepare paraffin sections. Four-micrometer consecutive tissue sections were cut from each arrayed paraffin block and prepared on pathological slides. The slices were stained by alcian blue/orange G (ABOG) or tartrate-resistant acid phosphatase (TRAP) for histologic analysis. The inflamation area, cartilage area, osteoclast number at ankle joints, and astragalus bone area of the ankle joint of mice in 3 groups were statistically analyzed. Slides were scanned using the Olympus VS120-S5-E whole-slide imaging system . All images were analyzed using the Olympus OlyVIA software .The number of lymphatic vessels was counted by immunofluorescence staining. The tissue sections were added with trypsin and repaired in 37°C incubators for 15 min, blocked by 4% Bovine Serum Albumin (BSA) for another 1 h. Primary FITCα-SMA antibody  and hamster monoclonal antimouse podoplanin antibody  incubate overnight at 4°C. The second Goat anti-hamster podoplanin  was incubated for another 1 h and immunofluorescence staining slides were scanned using an Olympus VS-120 whole-slide imaging system.Whole-mount staining of the back skin of the foot was stained to analyze the percentage of smooth muscle cell (SMC) coverage. The skin of the back of the foot was obtained when mice were sacrificed. We removed fat and other connective tissue, and then soaked the kin in 10% formalin at 4°C overnight and then permeated with 0.5% Triton X. After blocking with 4% Bovine Serum Albumin (BSA) for another 1 h, the skin tissues were incubated with 1: 1,000 dilution of Podoplanin antibody  and 1:400 dilution of fluorescein isothiocyanate labeled anti-SMA antibody at 4°C overnight. The images were recorded by Olympus VS-120 software. The whole operation should avoid light. The calculation formula is as follows: SMC coverage percentage = (SMC coverage area/whole lymphatic vessel area) × 100%.The tight ankle joints were obtained when mice were sacrificed and then fixed in 10% formalin for 48 h, washed in phosphate-buffered saline (PBS) for 2 h, and then soaked in 75% ethanol, scanned by micro-CT system . The X-ray tube voltage was 55 kV and tube current 72 μA, with a voxel size of 10 µm. The cross-section images were realigned in 3D using the SCANCO proprietary software to observe the morphology of astragalus.Peripheral blood samples were collected from mice when they were sacrificed. Blood samples (1 ml each) were collected from mouse eyeballs and then centrifuged to extract the upper serum. The serum level of Inflammatory cytokines TNF-α  and IL-6  were detected in strict accordance with the instructions of the kit.2.Mouse Primary Lymphatic Endothelial Cells/C57-6092  were cultured in α-MEM  supplemented with 100 IU/ml of penicillin/streptomycin  and 10% heat-inactivated fetal bovine serum  and maintained at 37°C in a humidified atmosphere with 5% CO4 cells/ml seeded in a 96-well plate, then exposed at various concentrations of NG-R1 for 24 h. A total of 10 µl CCK-8 solution was added into each well for another 1 h at 37°C. The resulting optical density was detected at 450 nm by a microplate reader (Biotek).Cytotoxicity assay was measured by Cell Counting Kit-8 (Dojindo. Japan. cat. #CK04) assay according to the manufacturer’s instructions. LECs were adjusted at a density of 1 × 10Co-culture was performed with Transwell . LECs were seeded on chambers in 6-well plates for 24 h, and then pretreated with NG-R1  for 2 h, stimulated and then pretreated with NG-R1  for 2 h and stimulated with TNF-α, culture chambers with LECs were then transferred into another 6-well plate, which had already been coated with LSMCs for 2 days. After 24 h of co-culture, LSMCs were harvested for analysis of the expression level of functional muscle genes h1-calponin, smα22, SM myosin heavy chain, and smα2 mRNA.Total RNA was extracted using Trizol (Thermo.United States. cat. #15596018) reagent according to the manufacturer’s instructions and the cDNA obtained by using the PrimeScript TM RT kit (Taraka. Japan. cat. #RR037A) according to the manufacturer’s protocol. Real-time PCR analysis was performed using Hieff TM qPCR SYBR Green Master Mix (Taraka. Japan. cat. #1 1201ES08) reagents on q-PCR CFX96 machine (Bio-rad). The primers for qRT-PCR were shown in The protein was collected, and 30 µg of protein from each sample was separated by 15% SDSPAGE and transferred to polyvinylidene flfluoride membrane and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and transferred to a Polyvinylidene flfluoride (PVDF) membrane. After blocking with 5% bovine serum albumin in TBST at room temperature for 2 h, the membranes were incubated with the Phospho-IKKα (Ser176)/IKKβ (Ser177) Antibody (cat. # 2688), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (cat. #3033) overnight at 4°C. After washing with TBST for three times, the membranes were incubated with the corresponding horseradish peroxidase-labeled secondary antibodies (cat. #7074). Proteins were scanned using the ECL detection system. All the antibodies were purchased from Cell SignalingTechnology .p < 0.05 were considered statistically significant.Statistical analysis was performed by Graphpad Prism (Version 8.0). Data are presented as means ± standard error of mean (SEM). One-way ANOVA followed by Dunnett’s t-test was used to determine differences between groups. As to 3 groups with different time points, we applied two-way ANOVA (or nonparametric) comparison. To establish whether NG-R1 reduced inflammation, we used TNF-Tg mice model and treated with NG-R1 for 8 weeks . As for A histopathological evaluation of the ankle joints was performed to examine the degrees of inflammatory damage. Histological examination and histomorphometric analysis showed that ankle joints from the model group had severe synovial inflammatory hyperplasia, cartilage loss, bone destruction, and greater osteoclasts numbers around the astragalus. ABOG staining analysis showed that NG-R1 treatment group had conspicuous reduced synovial inflammation, bone erosion, and cartilage destruction . TRAP stOur previous studies demonstrated that sufficient lymph drainage helps to improve joint inflammation . We nextTo prove whether the mechanism of NG-R1 improving lymphatic function is related to promoting lymphatic structural recovery and increasing the number of lymphatic vessels, we performed Whole Mount staining and immunofluorescence staining. Whole Mount staining revealed that the structure of the lymphatic vessel was significant destruction and the area covered by lymphatic smooth muscle was reduced in the model group . The areTo further verify the effect of NG-R1 on lymphatic smooth muscle cell function, we simulated the lymphatic structure and established a coculture system. LECs with or without NG-R1 pretreatment were stimulated with TNF-α and then co-cultured with LSMCs via transwell for 24 h. The results demonstrated that paracrine factors from the TNF-α stimulated LECs reduced the expression levels of muscle-related genes, including h1-calponin, smα22, smooth muscle myosin heavy chain, and smα2 in the LSMCs, which is prevented by NG-R1 . NG-R1 iWe investigated the CCK-8 kit to assess the potential cytotoxicity of NG-R1, and we found that NG-R1 did not affect cell viability on LECs after incubation 24 h even at a concentration of 100 µM . TherefoIt has been demonstrated that the NF-κB signaling pathway is a crucial inflammatory pathway involved in the development of rheumatoid arthritis. Thus, we examined whether NG-R1 regulates the activation of NF-κB signaling in LECs cells stimulated with TNF-α. We found that the phosphorylation of IKKα/β and p65 was markedly increased after being stimulated with TNF-α (20 ng/ml) for 15 min and 30 min. The phosphorylation of p65 was significantly inhibited in TNF-α-induced cells by treatment of NG-R1 (50 µM). However, it did not affect the phosphorylation of IKKα/β . In addiNG-R1 exhibits a pharmacological activity against excessive inflammation, but its mechanism of action has not been studied in RA. Here, our study is the first time to investigate the anti-inflammatory effect of NG-R1 in RA as well as elucidate its possible mechanism by which NG-R1 alleviates RA symptoms. Our current study demonstrated NG-R1 as a potential effective therapeutic medicine for the treatment of RA. We found that NG-R1 effectively improved inflammatory symptoms in the ankle joint of TNF-Tg mice by promoting the lymphatic drainage function while suppressing NF-κB signaling pathway-mediated production of proinflammatory effectors in LECs.in vivo.Notoginsenoside (NG-R1) is a novel triterpene saponin compound, one of the main bioactive ingredients from Panax notoginseng (PN) root, which is commonly used to treat cardiovascular diseases , neurodegenerative disease , diabetiThe lymphatic system plays an integral role in the development and progression of a range of disease conditions , which maintains extracellular fluid homeostasis as favorable, promotes metabolism, regulates immunity, and this also proves that it is possible to study the pathology and mechanism of the disease from the perspective of the lymphatic system . We have shown previously that TNF-Tg mice with severe arthritis typically have reduced or loss of lymphatic vessel contractions and decreased lymphatic flow from inflamed joints, but the mechanisms involved are unknown. Our previous studies showed that sufficient lymph drainage helps to improve joint inflammation, and drugs activating lymphatic function can attenuate synovial inflammation . In this study, we showed NG-R1 promoted lymphatic drainage function in TNF-Tg mice by accelerating lymphatic pulse frequency and lymphatic clearance. Next, we investigated the effect of NG-R1 on the structure and number of lymphatic vessels. Immunofluorescence staining results confirmed that NG-R1 did not affect the lymphatic vessel numbers in TNF-Tg mice, but it improved the destruction of lymphatic structures in the inflammatory state and increased the area covered by lymphatic smooth muscle.Our previous studies have confirmed the importance of lymphatic system in arthritis inflammation. In this study, as a common inflammatory inducer, TNF-α was applied to evoke extreme inflammation in LECs. TNF promoted the production of nitric oxide (NO) by LECs and caused LSMCs apoptosis, reduced expression of muscle functional genes, and LSMCs contribute to the dysfunction of synovial lymphatic vessels in inflammatory arthritis. Thus, we conducted the coculture system of LECs and LSMCs.The results demonstrated that NG-R1 increased the expression of lymphatic smooth muscle cell function marker geneh1-calponin, smα22, SM myosin heavy chain, and smα2 and promoted the contractile function of LSMCs and accelerated lymphatic drainage. NF-κB signaling pathway is one of the important transcriptional pathways in RA, which mainly regulates the level of pro-inflammatory mediators and induces the expression of IL-1, IL-6, and TNF-α and promotes the phosphorylation of p65 and IKKα/β in LECs. Our results show that NG-R1 dose-dependently inhibits TNF-α induced inflammatory cytokines TNF-α, IL-1β, IL-6, and iNOS production in LECs. NG-R1 repressed the phosphorylation of p65, and p65 nuclear accumulation relieved TNF-α-elicited inflammatory damages. However, NG-R1 without any effect on the phosphorylation of IKKα/β under the stimulation of TNF-α. Furthermore, inhibiting NF-κB signaling pathway in TNF-TG mice also inhibited the expression of serum inflammatory factors and improved synovial inflammation, cartilage erosion, and bone destruction of the ankle joint. These have led us to speculate that NG-R1 may play an anti-inflammatory role in RA. In future work, we also plan to continue to conduct experiments to explore NF-κB signal pathways by adding inhibitors or activators.In summary, using TNF-Tg mice as a model of chronic inflammatory arthritis, we reported for the first time that NG-R1 promoted lymphatic drainage function to ameliorate rheumatoid arthritis by suppressing the NF-κB signaling pathway. Thus, inhibiting inflammatory cytokines production and improvement of the lymphatic vessel draining function together represent a potential therapeutic strategy for RA. Meanwhile, it also represents that NG-R1 is a new potential treatment for rheumatoid arthritis.


Certain innovative technologies applied to medical product development require novel evaluation approaches and/or regulations. Horizon scanning for such technologies will help regulators prepare, allowing earlier access to the product for patients and an improved benefit/risk ratio. This study investigates whether citation network analysis and text mining of scientific papers could be a tool for horizon scanning in the field of immunology, which has developed over a long period, and attempts to grasp the latest research trends. As the result of the analysis, the academic landscape of the immunology field was identified by classifying 90,450 papers (obtained from PubMED) containing the keyword “immune* and t lymph*” into 38 clusters. The clustering was indicative of the research landscape of the immunology field. To confirm this, immune checkpoint inhibitors were used as a retrospective test topic of therapeutics with new mechanisms of action. Retrospective clustering around immune checkpoint inhibitors was found, supporting this approach. The analysis of the research trends over the last 3 to 5 years in this field revealed several candidate topics, including ARID1A gene mutation, CD300e, and tissue resident memory T cells, which shows notable progress and should be monitored for future possible product development. Our results have demonstrated the possibility that citation network analysis and text mining of scientific papers can be a useful objective tool for horizon scanning of life science fields such as immunology.The online version contains supplementary material available at 10.1007/s43441-021-00351-3. The application of innovative technologies that would lead to a product of novel modality or mechanism of action is expected to be a potential new therapeutic or diagnostic tool for diseases. However, there may be cases where the application of conventional development and evaluation concepts, regulatory frameworks, or both to innovative technologies are inappropriate. Therefore, early identification of innovative technologies with potential applications to medical products through horizon scanning would encourage regulatory authorities to establish new approaches to assess their quality, efficacy, and safety to advise developers and revise their regulations as needed. This could also contribute to timely patient access and improve the benefit-to-risk ratio of the product .Traditionally, horizon scanning has been predominantly conducted in Europe for policy-making, scientific research funding, and healthcare budgeting purposes, by surveying the Internet, government, international organizations and companies, databases, and journals using the Delphi method , 3. The Hines et al. reported that in the medical and healthcare field, most horizon scanning methods used were manual or semi-automated, with relatively few automated aspects. It is difficult to understand the whole picture of the extremely large and fragmented results of research and technological development. It might also be inappropriate to narrow the scope of consideration based solely on experts’ opinions, since information from experts might be subjective and the outcome depends on the choice of the expert. To solve this challenge, a computer-based approach can be used to complement the expert-based approach, as it fits the scale of the information , 8. In pIn this study, we focused on T cell immunity, because the research and the market has grown rapidly to be one of the major fields for developing pharmaceuticals, but the research history of the field shows an unexpected complexity which is considered to be a feature significantly different from other scientific fields such as artificial intelligence and nanocarbon. We explored if Sakata’s method is able to apply to immunology field focusing on immune checkpoint inhibitors as a retrospective example with new mechanisms of action and identified new topics in this field optimizing the horizon scanning method according to the target field.To select queries for citation network analysis and extract key articles to track the R&D history of immune checkpoint inhibitors, we selected the six key articles –24 shownThe keywords appearing in the key articles were used as search queries for paper titles, abstracts, author keywords, and keywords to extract papers, which included citation information for citation network analysis in the Web of Science literature database Web of Science Core Correction  and PubMed . To avoid subjectively narrowing down the papers for analysis and to include as many key articles as possible, we used “immune*” AND “t lymph*” as queries for both PubMED and Web of Science. We obtained 132,433 papers from the PubMed search, of which 90,450 papers (68.3%) formed a citation network containing five of six key articles. From the WoS search, 41,880 papers were obtained, of which 37,297 papers (89.1%) formed a citation network, but only one key article was included. We used only papers obtained from PubMed, which are considered to cover more papers than WoS on the targeted immune checkpoint inhibitors for the following analysis of citation networks and text mining.Q value) maximization method and are densely aggregated (Louvain method) [Q is an evaluation function of the degree of coupling within a cluster and between clusters, and is given as follows:δ-function δ is 1 if In this study, a citation network was converted into an unweighted network with papers as nodes and citation relationships as links. Papers with no citations as the largest component were considered digressional and were ignored in this study  , 28. TheThe cluster No. labels are assigned numbers based on the number of papers included in the cluster. The characteristics of each cluster were confirmed by extracting a summary/abstract of frequently cited academic papers in the cluster and the characteristic keywords in the cluster.In addition, we computed the term frequency-inverse cluster frequency (TF-ICF) to extract the characteristic keywords that were mechanically extracted by text mining with high TFCIF Table  of each To confirm the trends in the research field, the mean and median year of publication of papers in each cluster were extracted, as well as information on journals, authors, and affiliated institutions.After clustering the network, visualization was converted to intuitively infer relationships among these clusters. We use a large graph layout (LGL), which is based on a force-direct layout algorithm , 30. ThiWe analyzed a citation network of papers obtained from PubMed, and 38 clusters were formed. Table We selected the six key articles shown in Table It is suggested that Cluster 2 might be related to cancer immunity.In order to validate the method by confirming whether it can visualize the research progress and demonstrate the overall research landscape of the immune field, we assessed the transition of research on immune checkpoint inhibitors by analyzing papers published up to each year and identified the cluster containing the key articles in Table In this method, clusters are constructed based on papers with related citations; therefore, papers with several common citations, that is, papers that are likely to have similar content, are placed in the same cluster. The key articles in Table To detect the latest research trends in T lymphocyte immunology in this area, we reanalyzed ‘young’ clusters, which contains more papers published recently. The cluster often includes papers with broadly related topics, and sub-clustering may reveal individual topics; therefore, we reanalyzed clusters 1 to 5, which are the top five clusters with the most papers included as well as clusters 15, 16, 17, and 19, which were the top five in terms of the percentage of papers published in the last 3 or 5 years among the papers in each cluster Table , it is sIn this study, we applied citation network analysis and citation network analysis of bibliographic information in the field of immunology, which takes a rather long period to progress research due to the complexity of in vivo molecular interactions. For the immune checkpoints, CTLA-4 and PD-1 were identified in 1987  and 1992We were able to grasp the academic landscape of the field of immunology thorough the analysis as shown in Table Inhibitory antibodies against CTLA-4 and PD-1 were first reported to have anti-tumor effects in preclinical studies in 1996  and 2002The results of our analysis during this period are shown in Fig. Figure Next, we investigated recent research trends in this field Table . Owing t. [Thus, this study shows that citation network analysis and text mining using the methods of Sakata et al.  can be u. , 47, butThis study is the first to show that this method can be used appropriately as a tool for horizon scanning in the medical field. Because it is difficult to extract a limited number of novel topics that may affect pharmaceutical regulations from a vast amount of information on a human basis, it is reasonable and appropriate to use a computer-based method such as the method used in this study as a primary screening .We assume regulators as the end users of this method, which offers a new tool for horizon scanning to extract new topics from a huge database leading to the development of guidance and revisions to pharmaceutical regulations. This method might be applied to other government activities, such as HTA, research funding, and the business field.There may be limitations to our approach. One is the time delay until the publication of the research results, because most recently published papers without citation relationships cannot be included in this analysis method. However, we consider its impact on horizon scanning, targeting the stage before clinical development. Since it generally takes a few years from when the data have a high possibility of product development, non-clinical proof of concept, for example, to the start of clinical trials, we consider that our method can predict technologies that may lead to clinical development based on mid- to long-term research trends, even taking into account the time delay for publication. Another limitation is the necessity of the evaluation by experts in multiple aspects, such as expected medical positioning and patent information in order to achieve the purpose of horizon scanning, because our proposed method is based on the analysis of bibliographic information. In addition, we have not confirmed whether this method can be applied to all research domains. Hence, it may be necessary to consider an appropriate strategy for the utilization of this method in each field.data not shown). This suggests that the bibliographic database for analysis needs to be chosen according to the target field.We also suggest that selection of a bibliographic database is critical. We extracted scientific papers for citation network analysis from PubMed, which consists of more than 30 million citations to biomedical papers from MEDLINE, life science journals, and online books .We also Other methods are being explored to predict the future research activity of a certain topic. For example, in addition to cluster analysis, budding prediction was proposed to predict the growth of identified high-profile papers in the fields of solar cells and nanocarbons , 18.Most of the other horizon scanning activities in the health science field have been limited to HTA including IHSI activity focus on post-clinical and pre-marketing technologies. Our study is new in that it focuses on technologies that would be in clinical development in a relatively short period of time, with the aim of identifying novel technologies that could have regulatory implications at an early stage. Mechanical analysis using citation networks and text mining is used in the science map reported by the Japanese National Institute of Science and Technology Policy (NISTEP). However, they aim to obtain a bird's eye view of the entire scientific field by the top 1% of papers in terms of citations using "co-citation". Sakata’s method seems to be more suitable for our purpose since the method applies ‘direct citation’, which has been reported to be the most appropriate for obtaining leading-edge information on trends, to all papers extracted from the database .Thus, this study demonstrates that the proposed method of horizon scanning targeting technologies prior to clinical development based on citation network analysis and text mining is unique.. can objectively identify the emergence of new topics and their development through periodic analysis. We identified several candidate topics such as iRGD, ARID1A, and CD300e, for which tracking future research progress and potential applications to pharmaceuticals are recommended. The obtained results establish an efficient primary screening tool for horizon scanning procedure that enables regulators to prepare for new technologies, potentially benefiting patients through earlier access to the innovative products.This study showed that the citation network analysis and text mining of scientific papers by the methods of Sakata et alSupplementary file1 (PDF 187 KB)Supplementary file2 (PDF 208 KB)Supplementary file3 (PDF 300 KB)Below is the link to the electronic supplementary material.


Aims: To evaluate the interrelation between neutrophil to lymphocyte ratio (NLR) coupled with gene signatures, inflammation, and diastolic dysfunction in patients with heart failure (HF) with preserved ejection fraction (HFpEF).Methods: The clinical profile of 172 patients with HFpEF (EF ≥ 50%) and 173 non-HF control individuals was analyzed retrospectively. The association between NLR and HFpEF and the predictive performance of NLR for HFpEF were assessed by the binary logistic regression analysis and the receiver operating characteristic curve (ROC). Multivariate linear regression models further examined the associations between NLR and high-sensitivity C-reactive protein (hs-CRP), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), and average septal-lateral E/e', respectively. The freshly isolated neutrophils from 30 HFpEF patients and 42 non-HF controls were subjected to transcriptomic profiling. The biomarkers related to neutrophil activation and inflammation were detected in serum samples.Results: The HFpEF patients in Southeast China were lean and had comorbidity burden and worse cardiac structure/function. Compared with non-HF control individuals, HFpEF patients had a rise in NLR. NLR displayed an independent association with HFpEF  and it predicted HFpEF with the area under the ROC 0.796 . The positive associations between NLR and hs-CRP, NT-proBNP, and mitral E/e' were found in HFpEF patients. Moreover, patients had significantly elevated serum levels of neutrophil elastase and inflammatory biomarkers, both of which correlated with the mitral E/e' ratio. Finally, multiple molecules that drive neutrophil degranulation and inflammation, such as S100A8/A9/A12 and PADI4, were transcriptionally up-regulated in neutrophils of HFpEF patients.Conclusions: The high NLR coupled with transcriptional activation of neutrophils correlates with systemic inflammation and functional impairment in HFpEF patients, which may suggest a causative role of neutrophils in the pathogenesis of the disease. Heart failure (HF) with preserved ejection fraction (HFpEF) has conferred substantial morbidity and mortality on clinical patients. Its prevalence is increasing at an alarming rate, currently representing 50% of all HF worldwide . In contNeutrophils are the dominant type of leukocytes during acute inflammatory reactions. The emerging evidence that neutrophils contribute to the clinical manifestations of cardiovascular diseases has been well-discussed . In the It appears that neutrophils play significant roles in the pathogenic process of HFpEF. However, the pathological involvement of neutrophils in exacerbating the inflammation or functional impairment of HFpEF patients remains poorly understood. To this end, our study aimed to assess the interrelation between neutrophils coupled with transcriptomic profile, inflammatory biomarkers, and abnormal cardiac structure/function of clinical HFpEF patients.First, in retrospective analysis, the clinical data were obtained from 172 in-patients diagnosed with HFpEF (EF ≥50%) between January 2016 and December 2019 . PatientLaboratory variables, including complete blood cell counts, serum lipids, glucose, hemoglobin A1c (HbA1c), N-terminal prohormone of brain natriuretic peptide (NT-proBNP), were examined and documented. The NLR ratio was constructed as follows: NLR = neutrophil count to lymphocyte count.The circulating neutrophils were freshly isolated from eight HFpEF patients and 12 non-HF control individuals. The blood sample (2 mL) was carefully layered over Polymorphprep™ reagent . After centrifuging at 500 g for 35 min at room temperature, the plasma and mononuclear cells (upper band of cells) were removed, and neutrophils were harvested. After washing with Hepes-buffered saline [0.85% (w/v) NaCl], cell pellet was resuspended in ammonium chloride lysis buffer  to remove any residual erythrocyte contamination. Then cells were harvested by centrifugation and stored in TRIzol™ reagent  for subsequent RNA extraction.p-value  < = 0.05 was acceptable to indicate the gene expression with a significant difference. According to the results of differential gene detection, the R package heatmap was used to perform hierarchical clustering analysis on the union set differential genes. The Reactome enrichment was subsequently performed to investigate the molecular function and biological pathways that genes participate.RNA isolation and purification were performed using TRIzol-chloroform and RNeasy Mini Kit . The quality of RNA was checked with Agilent 2100 Bioanalyzer . The RNA-seq library was prepared by the Beijing Genomics Institute . Sequence reads were obtained using BGIseq500 (Illumina) and successfully mapped to the human genome . Read counts were normalized based on reads per kilobase million (RPKM). The DEseq2 method was used to filter differential genes . The adjThe serum samples were collected from 30 HFpEF patients and 42 non-HF control individuals. Biomarkers related to systemic inflammation , tissue remodeling [matrix metalloproteinase 9 (MMP9)], as well as neutrophil activation [neutrophil elastase (NE)] were examined by the enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions .t-test with Welch's correction. For all statistical analyses, p < 0.05 was considered to indicate statistical significance.In the cohort study, data are given as means and standard deviations (SD), medians and interquartile 25th and 75th percentiles (IQRs), or numbers and percentages, as appropriate. The statistical calculations were performed using IBM SPSS statistics software. Depending on the types of data, the non-parametric Mann-Whitney test or Fisher exact test for unpaired observations was applied for statistical comparison. Binary logistic regression analysis was carried out using HFpEF as the dependent variable to analyze the association between NLR and HFpEF after adjusting for potential confounders, including age, gender, body mass index (BMI), high-sensitivity CRP (hs-CRP), and diabetes. The crude and adjusted odds ratios (OR) with 95% confidence intervals (CI) were calculated. Receiver operating characteristic (ROC) curves with area under the curve (AUC) were calculated to determine discriminative ability. The partial Pearson or the Spearman correlations were computed to describe the relationship between variables of interest after values were logarithmically transformed. Subsequently, the multivariate linear regression analysis was conducted to identify factors associated with hs-CRP, NT-proBNP, and average septal-lateral E/e', respectively. In the transcriptomic sequencing study, the fold changes of FPKM of interested genes were calculated and compared between the two groups. Independent samples were compared by a two-tailed unpaired 2. The majority of HFpEF patients had severe cardiac function impairment. Approximately 90% of patients were classified in NYHA III to IV, together with a significant elevation of NT-proBNP in patients' blood. Besides hypertension, comorbid arrhythmia and coronary vascular disease were frequently present in HFpEF patients. Of note, we found diabetes was more common in the HFpEF group . Compared with non-HF individuals, a greater proportion of HFpEF patients were on an antiplatelet, beta-blocker, diuretic, and angiotensin-converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARBs) therapy. Echocardiography examination demonstrated that HFpEF patients had an overall prevalence of LV hypertrophy. Although HFpEF patients exhibited a preserved LV ejection fraction , the significantly increased average septal-lateral E/e' ratio, one of the echocardiographic markers of LV filling pressure   . Then, w< 0.001] .p < 0.001), NT-proBNP (p < 0.01), and mitral E/e' ratio (p < 0.05), respectively  was observed in HFpEF patients. By contrast, although MMP9 showed a correlation trend with both NE and E/e' ratio, it did not reach a statistical significance , a protein that critically regulates chromatin de-condensation and NETs formation  with relatively high abundance in neutrophils were up-regulated in the HFpEF group as well. However, their pathophysiological relevance with cellular activation remains obscure yet.We further characterized the transcriptional plasticity of neutrophils collected from non-HF controls and HFpEF patients. A total of 19,813 genes were successfully identified by RNA-sequencing. Among them, 134 genes were filtered with a significantly differential expression between the two groups . Concretpatients . S100A8/patients  or by mepatients . In addiry sites . Compareormation  24). Th. ThS100Ativation . FinallyThe main findings from the present HFpEF study were as follows: (1) the patients in Southeast China were lean and who had comorbidity burden and worse cardiac structure and function; (2) the high NLR was predictive to HFpEF and independently associated with hs-CRP, NT-proBNP, and mitral E/e' ratio; (3) the heightened serum NE levels correlated with the systemic inflammation and mitral E/e' ratio in HFpEF patients; (4) multiple molecules that drive neutrophil degranulation and inflammation were transcriptionally up-regulated in neutrophils of HFpEF patients.2) exhibit comorbidity-driven microvascular inflammation, HF severity, and fibrosis , inflammasome pathways, aging, stress, and disturbed lifestyle . We note program . Myocard program . Alternain vitro  database. The data are accessible via the SRA accession: PRJNA717666.The studies involving human participants were reviewed and approved by Research Ethics Committees of Shenzhen Second People's Hospital. The patients/participants provided their written informed consent to participate in this study.BB, HC, and YX were responsible for the study design and manuscript writing. BB, MC, LJ, and JX contributed to the acquisition and analysis of the data. All authors gave final approval and agreed to be accountable for all aspects of work to ensure integrity and accuracy.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.


Background: Left bundle branch area pacing (LBBAP) is a novel pacing modality with stable pacing parameters and a narrow-paced QRS duration. We compared heart failure (HF) hospitalization events and echocardiographic measures between LBBAP and right ventricular pacing (RVP) in patients with atrioventricular block (AVB).Methods and Results: This multicenter observational study prospectively recruited consecutive AVB patients requiring ventricular pacing in five centers if they received LBBAP or RVP and had left ventricular ejection fraction (LVEF) >50%. Data on electrocardiogram, pacing parameters, echocardiographic measurements, device complications, and clinical outcomes were collected at baseline and during follow-up. The primary outcome was first episode hospitalization for HF or upgrade to biventricular pacing. LBBAP was successful in 235 of 246 patients (95.5%), while 120 patients received RVP. During a mean of 11.4 ± 2.7 months of follow-up, the ventricular pacing burden was comparable (83.9 ± 35.1 vs. 85.7 ± 30.0%), while the mean LVEF differed significantly (62.6 ± 4.6 vs. 57.8 ± 11.4%) between the LBBAP and RVP groups. Patients with LBBAP had significantly lower occurrences of HF hospitalization and upgrading to biventricular pacing than patients with RVP , and differences in primary outcome between LBBAP and RVP were mainly observed in patients with ventricular pacing >40% or with baseline LVEF <60%. The primary outcome was independently associated with LBBAP , previous myocardial infarction , and baseline LVEF .Conclusion: Permanent LBBAP might reduce the risk of HF hospitalization or upgrade to biventricular pacing compared with RVP in AVB patients requiring a high burden of ventricular pacing.Clinical Trial Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03851315; URL: http://www.chictr.org.cn; Unique Identifier: ChiCTR2100043296. Some patients with advanced atrioventricular block (AVB) may be at high risk of pacing-induced heart failure (HF) because conventional right ventricular pacing (RVP) can result in left ventricular mechanical dyssynchrony and impaired cardiac function . A previLeft bundle branch area pacing (LBBAP) has emerged recently  as a newThis study was conducted at Fuwai Hospital, National Center for Cardiovascular Diseases, Beijing; Anzhen Hospital, Beijing; Sun Yat-sen Memorial Hospital, Guangzhou; the First Affiliated Hospital of Zhengzhou University; and the Second Hospital of Hebei Medical University. This prospective observational study was approved by the Institutional Review Board of all five hospitals in this study. All consecutive patients with AVB requiring ventricular pacing according to current guidelines were enrolled from 2019 if they signed written informed consent for an agreement of the implantation procedure and study analysis. Patients or the public were not involved in the design, conduct, reporting, or dissemination plans of our research.Patients with AVB recruited in this study were over 18 years old and had LVEF >50% at baseline. The pacing strategies were determined by operators as per the clinical practice at each hospital. The LBBAP group included all patients who attempted the LBBAP procedure, while the RVP group included patients undergoing RV apex or septum pacing. Patients were excluded if they (1) were younger than 18 years; (2) underwent pacemaker replacement or upgrading with existing leads; (3) had severe valvular diseases, congenital heart disease, or hypertrophic cardiomyopathy; (4) were diagnosed with acquired AVB after surgery for hypertrophic cardiomyopathy or other congenital heart diseases; (5) were diagnosed with persistent atrial fibrillation; and (6) were unavailable to be regularly followed up at the clinic visit for various reasons or to provide written informed consent .4−6. Ring pacing was tested to evaluate the lead depth in the interventricular septum. Measures of impedance and R wave amplitudes are helpful to prevent lead penetration into the LV. If LBBAP could not be successful after five attempts, the lead was screwed into the interventricular septum to achieve deep LV septal pacing. The RV lead was implanted at the RV apex or septum by a shaping stylet to achieve stable pacing parameters.The LBBAP procedure has been previously described in detail . During Successful LBBAP was confirmed per the previously published criteria , 9, 11: The clinic visit follow-up was performed every 6 months after pacemaker implantation in each hospital. Echocardiographic evaluations were conducted at baseline, 6 months, and 1 year after the procedure by using Vivid E9 systems . Left ventricular end-diastolic diameter (LVEDD) and LVEF were evaluated by the core lab that was blinded to the pacing parameter settings, and in cases of disagreement, a senior echocardiographer was invited to read the original data to reach an agreement. Biplane Simpson's method in two-dimensional transthoracic echocardiography was used for the evaluation of LVEF.The primary outcome was defined as a combined endpoint including the first episode of HF hospitalization or the need for upgrading to BiVP. The independent event committee adjudicated all events. HF hospitalization was identified if the patient presented to outpatients or emergency department visits or inpatient hospitalization with symptoms and signs consistent with HF and required diuretics and other therapy . The indications for requiring an upgrade to BiVP were according to current guidelines , includit-tests or Wilcoxon rank sum test. Nominal data are presented as frequencies and percentages and were compared by using the chi-square test or Fisher's exact test. Kaplan–Meier curves and univariate and multivariate Cox proportional hazard models were used to analyze the primary outcomes, and time censoring was determined by time to primary outcomes or time to last follow-up. All statistical tests were two-tailed. A P value of < 0.05 was considered significant.The statistical analyses were performed by SPSS version 24.0  and GraphPad Prism 5 . Continuous variables were summarized using the means and standard deviation or median and interquartile range and compared with two-tailed Student's P = 0.005). Baseline LVEF was also comparable between the LBBAP (61.7 ± 7.4)% and RVP (61.5 ± 6.4)% groups.A total of 366 consecutive patients were included. LBBAP was attempted in 246 patients, while 120 patients received RVP. As shown in Permanent LBBAP was successful in 235 of 246 patients (95.5%), with selective LBBP in 162 (68.9%) patients. As shown in P = 0.614). At the 1-year follow-up, the pacing threshold and sensing R wave amplitude were comparable between the two groups. The lower pacing impedance and narrower QRSd  remained in the LBBAP group.The mean follow-up duration was 11.4 ± 2.7 months. The complications in the LBBAP group were similar to those in the RVP group. Even though five patients (2.1%) suffered septal perforation during the procedure, the perforation did not cause any symptoms. Only one septal perforation occurred 2 h after the procedure and resulted in dislodgement and ventricular capture failure. After repositioning the pacing lead, most patients underwent successful LBBAP with uneventful recovery. Lead perforations or dislodgement was not found following hospital discharge. In the RVP group, apical perforation occurred in one patient, ventricular lead dislocation occurred in three patients during follow-up, and all patients underwent uneventful lead revision.P = 0.004) and LVEDD .Compared with baseline, patients with LBBAP had stable LVEF and slightly decreased LVEDD at the 1-year follow-up . In contP <0.001, P = 0.003) and a higher occurrence of an upgrade to BiVP (P = 0.027) in the RVP group than in the LBBAP group (P = 0.005). HF hospitalization and upgrading to BiVP were also associated with a history of previous myocardial infarction  and LVEF at baseline .The primary composite endpoint of HF hospitalization and upgrading to BiVP was 2.6% in the LBBAP group and 10.8% in the RVP group (AP group . In TablP = 0.005) (n = 150), the RVP group had significantly higher composite HF events than the LBBAP group    but not = 0.005) . The dif= 0.005) . However= 0.034) . In patiThis multicenter prospective study demonstrated that permanent LBBAP presented stable pacing parameters and procedural complications similar to RVP during a 1-year follow-up. In patients with normal cardiac function and a high burden of VP, LBBAP achieved preserved LVEF and reduced LVEDD, while RVP resulted in reduced LVEF and enlarged LVEDD. Patients with LBBAP had a significant reduction in HF hospitalization events (including upgrading to BiVP) compared with conventional RVP . The effect of LBBAP was seen predominantly in patients with VP >40%, patients with LVEF ≤ 60%, or patients with baseline organic cardiac disease or AF. LBBAP was an independent predictor for a reduced risk of HF hospitalization after adjustment for other risk factors.The detrimental effect of traditional RVP has been associated with an increased risk for HF hospitalization and mortality in patients with a high VP burden . A recenLBBAP can pace the conduction system beyond pathological or disease-vulnerable regions to produce nearly physiological ventricular capture. In recent studies, LBBAP generally achieves paced QRSd within 130 ms, mostly between 110 and 120 ms –20. ThisLBBAP could achieve LV synchrony and preserve LV function in bradycardia patients with normal cardiac function . A recenTo our knowledge, this is the first study comparing LV function and clinical outcomes between LBBAP and RVP in AVB patients with high VP burden and baseline narrow QRSd. The significant difference in LVEF and LVEDD between the LBBAP and RVP groups at 1-year follow-up verified the beneficial effect of LBBAP on cardiac function. Although patients with normal cardiac function usually have few clinical outcomes after receiving RVP, our study still observed a significant difference in HF hospitalization events between LBBAP and RVP. A high burden of VP > 40% has been recognized as a risk factor for HF events during long-term follow-up. Our subgroup results further indicated that LBBAP might provide an additional benefit of cardiac function in patients with VP > 40%, baseline decreased LVEF (<60%), or baseline organic cardiac disease or atrial fibrillation. This is also the first study reporting the occurrence of HF hospitalization in patients with a high burden of LBBAP. Six patients in the LBBAP group presented HF symptoms and relatively reduced LVEF (>50 and <60%), and they recovered well after receiving medical treatment, including oral diuretics and beta-blockers. No patients in the LBBAP group presented with indications for upgrading to BiVP. Our results together with previous studies indicate that LBBAP might effectively reduce the risk of HF hospitalization compared with RVP in patients with normal LV function and a high burden of VP.The main limitation of this study is the observational study design. The clinical homogeneity of patients could not be guaranteed between LBBAP and RVP. However, the higher prevalence of atrial fibrillation in the LBBAP group further demonstrated the potential benefit of LBBAP compared with RVP. Second, the relatively small sample size and the high percentage of RVAP in the RVP group might contribute to the difference in the clinical outcomes between RVP and LBBAP. Third, the clinical outcomes of all-cause death or cardiovascular death during longer follow-up may provide more solid evidence for the superiority of LBBAP. Therefore, future prospective randomized clinical trials with a large sample size are needed in patients with a high burden of VP.The results of this multicenter observational study indicate that LBBAP might be a preferable pacing modality to reduce potential HF events in patients requiring a high burden of VP compared with traditional RVP.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of Fuwai Hospital, Beijing; Institutional Review Board of Anzhen Hospital, Beijing; Institutional Review Board ofSun Yat-sen Memorial Hospital, Guangzhou; Institutional Review Board of the First Affiliated Hospital of Zhengzhou University, Zhengzhou and Institutional Review Board of the Second Hospital of Hebei Medical University, Hebei, China. The patients/participants provided their written informed consent to participate in this study.XF, XL, YC, and YW conceptualized and designed the research. XF, XL, JZ, CQ, HL, KP, ZW, ZL, and RX were responsible for the acquisition, analysis, and interpretation of data. XF had obtained funding and supervised the work. XF and XL drafted the manuscript. YY, YC, and YW critically revised the manuscript. All authors contributed to the article and approved the submitted version.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.