The process of electrospinning is over a century old, yet novel material and method achievements, and later the addition of nanomaterials in polymeric solutions, have spurred a significant increase in research innovations with several unique applications. Significant improvements have been achieved in the development of electrospun nanofibrous matrices, which include tailoring compositions of polymers with active agents, surface functionalization with nanoparticles, and encapsulation of functional materials within the nanofibers. Recently, sequentially combining fabrication of nanofibers with 3D printing was reported by our group and the synergistic process offers fiber membrane functionalities having the mechanical strength offered by 3D printed scaffolds. Recent developments in electrospun nanofibers are enumerated here with special emphasis on biomedical technologies, chemical and biological sensing, and energy harvesting aspects in the context of e-textile and tactile sensing. Energy harvesting offers significant advantages in many applications, such as biomedical technologies and critical infrastructure protection by using the concept of finite state machines and edge computing. Many other uses of devices using electrospun nanofibers, either as standalone or conjoined with 3D printed materials, are envisaged. The focus of this review is to highlight selected novel applications in biomedical technologies, chem.-bio sensing, and broadly in energy harvesting for use in internet of things (IoT) devices. The article concludes with a brief projection of the future direction of electrospun nanofibers, limitations, and how synergetic combination of the two processes will open pathways for future discoveries. One-dimensional nanomaterials in the form of nanofibers have a wide-ranging spectrum of research and commercial applications, due to their exceptional physicochemical properties and characteristics. Electrospinning is a relatively simple, yet versatile and promising method for the production of nanofibrous materials for a wide variety of applications ,5,6,7,8.These nanofibers with cross-sectional diameters ranging from tens to hundreds of nanometers possess high surface energy due to a large surface area. Furthermore, they can form networks of highly porous mesh with remarkable interconnectivity between their pores, making them attractive for a multitude of innovative applications. The materials for nanofibers include natural polymers, synthetic polymers, carbon-based, semiconductors, ceramers, and composites. A few basic configurations of electrospinning apparatus are shown in Electrospun nanofibers exhibit exceptional and unique properties including high extensibility and high mechanical strength. Electrospinning technology has gained enormous interest in recent years both as a research topic and in futuristic industrial fields. Tremendous efforts have been devoted to the development of electrospinning process setups, strategies for successful electrospinning, characterization of electrospun nanofibers, and exploring novel applications of electrospun nanofibers in different fields. Significant improvement has been achieved in the development of electrospun nanofibrous matrices, which includes tailoring compositions of polymers and active agents, surface functionalization with nanoparticles, and encapsulation of functional materials within the nanofibers. Electrospun nanofibrous materials have attracted significant attention, due to the flexibility of electrospun nanofibers, providing suitable mechanical property requirements and high surface area for a wide variety of applications. The unique properties of electrospun materials, including high surface-to-volume ratio, flexibility, high mechanical strength, high porosity, and adjustable nanofiber and pore size distribution, render them potential candidates in a wide range of applications in medical and engineering fields. Electrospinning is becoming more efficient and more specialized in order to produce particular fiber types with variable diameter and morphology, tunable alignment, patterned, and 3D structure. By controlling electrospinning polymer parameters, additives, and production strategies, it is possible to control nanofibrous structures, with specific properties for applications in tissue engineering and regenerative medicine, textiles, water treatment, sensors, and energy fields.Biomaterials in the form of coatings, particles, spheres, fibers, thin films, foams, and gels can be designed using many different conventional or modified techniques developed for product manufacturing in the biomedical field. The main feature in the development of biomaterials for biomedical applications is biocompatibility, and according to this feature, Bonferoni et al. divided biomaterials into four categories in their study. In this case, first-generation biomaterials were characterized as bioinert, second-generation biomaterials as absorbable or bioactive, third-generation materials as both absorbable and bioactive, and fourth-generation biomimetic materials . As the Scaffolds are biomaterials, as one of the fundamental components of tissue engineering, developed for the repair/restructuring of lost/damaged tissues as a result of any accident or disease, congenital anomalies, or trauma . In the Apart from the addition of bioactive compounds, electrospun fibers are also used in the design of multi-part scaffolds by producing gradient layers ,45,46 anA wound is defined as a disruption of skin integrity and normal functioning as a result of various diseases, traumas, burns, or illnesses. Especially in the case of chronic and deep wounds, it is important to cover and protect the wound with dressings and rebuild a temporary body barrier. Current wound dressings used for wound healing are cost-effective but have limited functionality to meet the needs of the existing market. For example, they cannot accommodate complex wound healing environments, so healing is slow, or they can adhere to the wound surface, which can cause additional damage and pain when they are replaced . To overIn addition to active ingredients that exhibit antibacterial properties, extracts of many different plant species have been used in the design of wound dressing materials. In the studies conducted in this context, the changes caused by herbal additives in the material are examined by revealing the physicochemical properties. The issues that need to be emphasized and developed are the diffusion of these bioactive components into the wound, release behavior, and release modeling. There is also a need to improve the effects of such additives on the mechanical properties of the material.Cancer is a difficult disease that affects millions of people worldwide, is a source of high stress, and has become the leading cause of death. Therefore, it is of great importance to investigate the formation, invasion, and metastasis of the malignant tumors that cause cancer. One of the most important steps taken in this context is in vitro cancer models that mimic the microenvironment where cancerous cells are found and a tumor develops . In vitrIn addition, the topography of fibers plays a significant role in mediating neoplastic invasion and metastasis. In a study by Ali et al., they evaluated the scaffolds prepared with PCL and PLA polymers to create a 3D model of colorectal adenocarcinoma. By changing the polymer type and fiber alignment, changes occurred in the mechanical properties of the scaffold, hydrophobic properties, pore size, and porosity. Although Caco-2 cells managed to colonize in each tumor model, an opposite trend of cell metabolic activity occurred in PLA and PCL scaffolds as a function of fiber alignment, increasing in PLA and decreasing in PCL scaffolds . Electrospun fibrous structures prepared based on different natural and synthetic polymers have been mostly used in breast cancer in vitro modeling studies. Studies conducted in this context include chitosan/PEO , collage®, Lupron Depot®, and Sandostatin LAR®.Drug delivery systems describe technologies and materials developed to deliver a therapeutically effective amount of drug to the damaged/diseased area. Electrospun nanofibers have emerged as promising materials for the construction of nanoscale drug delivery platforms because of their large surface-to-volume ratio, high drug loading capacity of up to 60%, nearly 100% encapsulation efficiency, ability to modulate drug release, and controllable surface modification ,78. The Although the word “drug” is used to express release here, the loading of synthetic, natural, and biologically active substances into electrospun nanofibers and examining the release of these substances are also considered in this category. Therefore, the release of active substances ranges from various drugs such as antibiotics, anti-inflammatory, antimicrobial, cardiovascular, antihistamine, gastrointestinal, palliative, and contraceptive to macromolecules such as proteins and DNA . PolymerDespite the great advantages of drug-loaded fibrous materials produced on the basis of electrospinning, the number of clinical studies reported in the literature is not yet sufficient. In electrospinning, toxic residues of the solvents chosen to dissolve the polymers may be trapped in the fibers and released from the fiber along with the drug during the release period. For this reason, there is a need to focus on solvent selection studies and develop new approaches that utilize the use of more environmentally friendly and green solvents such as water instead of aggressive and toxic ones .The electrospinning process is a unique method to produce nanofibers of varying porosity and thus highly effective areas with large surface activation energy for sensing applications. Several combinations of synthetic and natural polymers; polymer composites; and polymers impregnated with nanoparticles, carbon nanotubes, ceramers, and other compounds have been synthesized for specific applications . DependiElectrochemical biosensors are the best candidates for measuring specific biomarker concentrations in body fluids . To incrThe onset of mobile and pervasive computing; wireless networks; network appliances; flexible substrates; and e-textile and tactile sensors is enabling the development of systems on textiles or systems on fibers (SoFs). The ability to disperse ceramics, metal oxides, nanoparticles, and carbon nanotubes in polymer solutions and develop fibers, thereafter employing electrospinning, has produced new pathways for functional fibers, mentioned here as SoFs—that have electronics and interconnections woven into them. Such systems demonstrate incredible potential for the development of portable functional devices such as fibers embedded with data communications systems, nanogenerators for energy harvesting, health and biomarkers monitoring, personal fitness, defense systems, mobile computing, targeted and time-released delivery of vaccines, protective armor, chemical-biological sensors, radio frequency identification (RFID) devices, Global Positioning System (GPS), and interactive geographical information systems (GIS). Additive processing technology offers the possibility for the development of systems with physical flexibility and size unachievable with current and conventional advanced manufacturing technology ,101. RecDue to the highly interconnected nature of our environment, there has been a growing need for ubiquitous sensing for enhanced situational awareness. One of the primary needs for ubiquitous sensing is to power/activate these devices in a standalone mode since a distributed power grid may not have the power option in the field for mobile, remote, and free-standing applications. Several sensors based on the piezoelectric effect offer the advantage of self-powered devices, however, have so far been limited to bulky and inflexible sensors. Recent interest in nanofibers doped with poly(vinylidene fluoride) (PVDF) and lead zirconate titanate (PZT) and PZT doped with different lanthanides inorganic fillers  have dem3), and trifloroethylelene (TrFE) have been experimented with to enhance performance figures of merit [PVDF is ideal as a sensing element for tactile sensing due to its low cost, flexibility, and biocompatibility. PVDF also possesses good thermal stability, low permittivity, and wide frequency response, and has excellent thin-film-forming properties . PVDF haof merit . These eij), viz. lead zirconate titanate (PZT), lanthanum-substituted PZT, lithium niobate (LiNbO3), and lead magnesium niobate-lead titanate (PMN-PT). As biomedical sensors, PVDF-based electrospun fibers are highly suitable for piezoelectric sensor applications due to their high piezoelectric, pyroelectric, and ferroelectric properties with high mechanical strength, high thermal stability, superior flexibility, and excellent biocompatibility . HoweverPVDF-based sensors/energy harvesters show promise for the early evaluation and prevention of cardiovascular diseases and respiratory disorders, as well as many pressure-related acute and chronic diseases . In receResearch on electrospun nanofibers is growing at a tremendous pace, especially due to the numerous advantages and benefits that nanofibers have to offer in various fields which include tissue engineering, wound dressing, drug delivery, biomedical engineering, energy harvesting, biosensors, and tactile sensing applications. The development in electrospinning technologies and combination of electrospinning with additive manufacturing techniques, such as 3D/4D printing, can lead to new strategic applications of this technology in the forthcoming years. Combining the electrospinning fabrication method with various types of materials including synthetic and natural polymers to fabricate tissue engineering scaffolds, biosensors, energy harvesting devices, and sensors could pave new paths and new research directions and industrial applications. Electrospun nanofibers with improved mechanical and functional properties can be manufactured by combining nanoparticles, active agents, and functional materials. However, although electrospinning technology has been greatly developed and received attention in the past two decades, there are still certain challenges with perfectly controlling the process parameters to produce nanofibrous materials with the same features consistently and improving the large-scale production speed of electrospinning for commercialization. More specifically, controlling porosity presents a challenge. Addition of materials to modify the fiber characteristics for desired applications presents a challenge as well. With the advent of finite state machine configuration, the use of nanogenerators in tactile sensors will play a major role in prosthetics and other functions that relate to monitoring body vitals remotely. Several applications in critical infrastructure are also possible and are currently being investigated. The application of electrospinning technology in the field of human health should be expanded to have a clear and deep understanding. The drug delivery systems produced by electrospinning to provide desired delivery profiles should focus on applying clinical trials to demonstrate effective treatments. Lastly, our efforts are towards a more sustainable future by taking into account life cycle of micro/nano particles  and plas


Undaria pinnatifida  was performed’ more than 900 molecular species were identified in 12 polar lipids and 1 neutral lipid using HPLC/MS-MS. The seasonal changes of U. pinnatifida lipidome were determined. It was shown that acclimatization to winter and spring was accompanied by an increase in the unsaturation of both polar and neutral lipids. In autumn and summer, on the contrary, the contents of more saturated molecular species of all lipid classes increased. Based on the data obtained, a scheme for the polar and neutral lipid synthesis in brown algae was proposed. In addition, the influence of infection with the brown filamentous endophyte Laminariocolax aecidioides  on U. pinnatifida lipidome was studied. It was found that infection has the most noticeable effect on the molecular species composition of triacylglycerides, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylhydroxyethylglycine of the host macrophyte. In infected samples of algae, changes in the composition of triacylglycerides were revealed both in areas with the presence of an endophyte and in adjacent intact tissues, which may indicate the occurrence of a secondary infection.An analysis of the lipidome of the brown alga  Undaria pinnatifida (Harvey) Suringar, 1873  is an annual brown macrophyte growing in littoral and sublittoral zones at a depth of 1–15 m. In many Asian countries, including Japan, China and Korea, these algae are cultivated and used as a food additive + ions have also been used to separate DGTS and DGTA molecular species according to the fragmentation reactions described by Li et al., 2017 [4]+ ions; the sn-positions of acyl groups were determined by fragmentation at 28 eV collision energy [The MS parameters were as follows: the temperatures of the interface, heat block, and desolvatation line were 300, 400, and 250 °C, respectively; the flow rates of drying gas  containing 100 mM formic acid and 40 mM ammonium hydroxide; 0.1 μL of lipid extract with a concentration of 1 mg/mL was injected. For the detection of lipid classes, the same mass spectrometer parameters were used as for the molecular species analysis. Individual lipid classes isolated from U. pinnatifida extracts were used as standards for external calibration. For triglyceride detection, a total ion current in the range of 700–868 + 880–1100 m/s was used, since pigments interfered in the range of 868–880 m/z.Quantitative analysis of lipid classes was performed using HPLC-MS. An Ascentis Si column   in HILIC mode at 40 °C was used. A binary high-pressure gradient was used : 0 min , 2.5 min , 2.99 min (0.2), 3 min (0.15), 5 min , 12 min (40%), 16–19.99 min , 20–21.49 min , 21.5–24.99 min , 25–26 min , mixer volume—40 µL. Eluent A was acetonitrile containing 50 mM formic acid, eluent B was acetonitrile:water . All values were presented as mean ± standard deviation for triplicate. The data were assessed statistically by one-way ANOVA and Tukey HSD test for a posteriori comparison, as well as by Student’s U. pinnatifida was characterized. Based on the data obtained, the biosynthesis pathways of various lipid molecular species in brown algae were determined.In this work, the complete lipidome of the brown alga U. pinnatifida have been established. It was shown that acclimatization to winter and spring is accompanied by an increase in the most unsaturated molecular species of plastidic lipids—MGDG and DGDG containing 18:4 and 20:5, PG containing C18-PUFA, as well as extraplastidic lipids PE, PC, and PHEG containing 20:5. In autumn and summer, the contents of less unsaturated MGDG and DGDG molecular species with 18:3 and 20:4, as well as PG and SQDG with SFAs and PE, PC, and PHEG containing 20:4, were increased. During the year, changes were also observed in the compositions of storage lipids in alga. In winter, ER-derived molecular species of TAG with PUFAs accumulated, while in spring, plastid- and ER-derived molecular species accumulated, representing combinations of SFAs, MUFAs, and PUFAs, and in autumn and summer, plastid- and ER-derived molecular species with SFAs and MUFAs did.The main molecular species of structural lipids involved in the seasonal adaptation of U. pinnatifida is often infected with the brown filamentous endophyte Laminariocolax aecidioides. We studied the influence of endophytic infection on the U. pinnatifida lipidome. In infected sections of the algal blade, the proportion of structural lipids, such as PG, PE, PC, and PHEG, containing PUFA was increased, which may be a reaction in response to mechanical stress caused by the growth of endophytic filaments inside the host macrophyte tissue. In infected, as well as adjacent intact tissue sections of the U. pinnatifida blade in the contents of TAG molecular species containing SFAs and MUFAs were increased, which may indicate the occurrence of a secondary infection.Under natural conditions,


The whole-examination effective dose was slightly lower for Scenario 1 . Notably, Scenario 1 resulted in a significantly lower radiation dose for sequential scans and obese patients. Only high-pitch spiral CCTA showed dose reduction in Scenario 2. Conclusions: Using CAS-CT for planning prospectively ECG-triggered CCTA reduced the overall radiation dose administered compared to scout view planning without CAS-CT, except for high-pitch spiral CCTA, where a slightly opposite effect was observed.Background: This study investigates whether the scan length adjustment of prospectively ECG-triggered coronary CT angiography (CCTA) using calcium-scoring CT (CAS-CT) images can reduce overall radiation doses. Methods: A retrospective analysis was conducted on 182 patients who underwent CAS-CT and prospectively ECG-triggered CCTA using a second-generation Dual-Source CT scanner. CCTA planning was based on CAS-CT images, for which simulated scout view planning was performed for comparison. Effective doses were compared between two scenarios: Scenario 1—CAS-CT-derived CCTA + CAS-CT and Scenario 2—scout-view-derived CCTA without CAS-CT. Dose differences were further analyzed with respect to scan mode and body mass index. Results: Planning CCTA using CAS-CT led to a shorter scan length than planning via scout view (114.3 ± 9.7 mm vs. 133.7 ± 13.2 mm,  Cardiovascular disease is the leading cause of mortality among adults worldwide . Early dDespite recent advancements in CCTA, concerns remain regarding radiation exposure. The Society of Cardiovascular CT has published guidelines for optimized CCTA acquisitions to minimize radiation exposure while maintaining diagnostic accuracy . Some suWith the emergence of second-generation Dual Source CT technology (DSCT), two novel, low-dose, prospectively ECG-triggered scan modes have been made available: sequential (“step and shoot”) and high-pitch spiral (“flash”) CCTA . As retrThus, our study aims to examine the influence of the scan length adjustment of low-dose, prospectively ECG-triggered CCTA using CAS-CT and determine whether the omission of CAS-CT represents a viable strategy for reducing the radiation dose of the whole examination.Approval was obtained on 11 March 2021 (No. 21-1060) by the institutional review board of the University of Freiburg Medical Center, and the need for informed consent was waived. Between January 2020 and September 2020, consecutive patients clinically referred for CCTA were retrospectively included in the study group. The following data on patient characteristics were recorded: gender, age, height, weight, clinical symptoms, pretest probability for CAD, blood pressure, and heart rate. Exclusion criteria included a deviating or incomplete examination protocol, scan abortion, and an already-known CAD. All CT examinations were conducted using a 2nd-generation DSCT scanner . Initially, an anterior–posterior scout view of the chest was obtained. The craniocaudal extension of CAS-CT was then planned using the scout view, with an upper limit of 1 cm below the carina and a lower limit of the apex cordis. Non-contrast CAS-CT was performed using a low-dose, high-pitch scan mode with the following parameters: tube voltage 120 kV, automated tube current modulation (CARE Dose mAs at a reference of 80 mAs), detector collimation of 2 × 128 × 0.6 mm, gantry rotation time of 280 ms, a pitch of 3.2, and a matrix size of 512 × 512. Images were acquired at 60% of the R-R interval during an inspiratory breath-hold, for which patients were trained by a doctor before each scan. Images were transferred with a default mediastinal window setting, a slice thickness of 3.0 mm, and a field of view restricted to the heart, and a sharp reconstruction kernel was applied (b36f). After CAS-CT, a beta-blocker  was administered intravenously if necessary, and patients received a single dose of 5.0 mg of isosorbide-dinitrate if there were no contraindications. The planning of the craniocaudal extension for CCTA was carried out using transversal CAS-CT images. To achieve this, one radiologist and one expert technician identified the table position of the highest point of the coronary tree, primarily the proximal left anterior descending artery, as well as the apex cordis by consensus. An additional 1 cm was added to these landmarks for the extension in order to account for variability in patient breathing. A test-bolus preceded CCTA to determine the time delay until maximal aortic contrast enhancement; accordingly, a region of interest was placed in the aortic root, and consecutive image acquisition started 10 s after intravenous injection of 10 mL of iodinated contrast material  followed by 60 mL using a dual-syringe power injector. The time to attenuation peak was measured, and 4 s were added to the time until peak in sequential scan mode and 5 s in high-pitch spiral scan mode to determine the CCTA scan delay . For CCTA, 60-72 mL of contrast material  was injected, followed by a 60 mL saline flush (injection rate: 6 mL/s). A patient-specific simple score, calculated based on our institutional standards of procedure for CCTA acquisition , was useWhen a sequential scan was applied, images were reconstructed in the automatically determined best diastolic or, if applicable, best systolic phase and in a multiphase dataset with 5% steps R-R interval . All CCTTwo radiologists with 4 (M.T.H.) and 9 years (M.S.) of experience in cardiovascular CT analyzed all images by consensus on a dedicated workstation . They were blinded to clinical history and prior imaging examinations. Any disagreements were resolved by involving a third radiologist with 15 years of experience in cardiovascular CT (T.K.). Image quality was assessed with respect to the coronary tree’s vessel sharpness, movement artifacts, and contrast attenuation. A five-point Likert scale was applied for image quality assessment, whose scoring details are as follows: 1 = “non-diagnostic” (extensive artifacts and vessel deformation), 2 = “fair” (many artifacts present yet determined diagnostic by consensus), 3 = “moderate” (blurred vessel contours and numerous artifacts), 4 = “good” (slight radiating artifacts), and 5 = “excellent” . An example is provided in The craniocaudal extension of CAS-CT was then planned from the anteroposterior scout view, with an upper limit of 1 cm below the carina and a lower limit of the apex cordis. The scan length of CAS-CT and the scan length of CCTA were determined by consensus involving one radiologist and one expert technician. In this process, the table position of the first image above the most cranial part of the coronary tree and the first image below the cardiac apex were noted. CCTA was planned by adding 1 cm to the upper and 1 cm to the lower cardiac border on CAS. The scan length of the simulated scout-view-based CCTA was assumed to be equal to the CAS-CT. Thus, the table positions of the uppermost and lowest axial images of calcium scoring were noted and used to calculate the scout-view-derived scan length of CCTA, as previously described . −1 × cm−1) between both sexes through Monte Carlo simulations  mGy*cm vs. 198 [94–345] mGy*cm (n = 150), Scenario 1 achieved a radiation dose reduction of 5.9%, i.e., 214 [138–359] mGy*cm vs. 227 [141–385] mGy*cm (p < 0.001). With ECG-padding of 20% or more of the R–R interval applied to sequential CCTA (n = 99), Scenario 1 resulted in a radiation dose reduction of 11%, i.e., 241 [164–431] mGy*cm vs. 269 [188–464] mGy*cm (p < 0.001). In contrast, for high-pitch spiral CCTA (n = 32), Scenario 2 demonstrated a lower radiation dose than Scenario 1, namely, 48 [40–80] mGy*cm vs. 57 [48–88] (p < 0.001). The results are summarized in n = 45)  or when a tube voltage of 120 kV was applied, namely, 359 [229–490] mGy*cm vs. 373 [222–527] mGy*cm (p < 0.001).When analyzing the subgroups stratified by specific prospective ECG-triggered CCTA scan protocols, it was observed that the differences between Scenario 1 and Scenario 2 were more pronounced. In sequential CCTA  consistently planning the scan range of CCTA using axial non-contrast CAS-CT images results in a shorter scan length than planning using a scout view. (II) The whole-examination radiation dose of prospective ECG-triggered CCTA planned via CAS-CT is slightly lower than CCTA planned using scout view, even if no CAS-CT is acquired in the latter scenario. (III) The CAS-CT planning of prospective ECG-triggered CCTA results in a radiation dose salvage compared to scout view planning when individual factors  or technical factors  contributing to a higher overall radiation dose are present. (IV) Omitting CAS-CT to lower the radiation dose of cCTA is not a reasonable strategy because the potential to reduce the CCTA scan range and, consequently, the radiation dose cannot be exploited. (V) In this regard, the acquisition of only low-dose high-pitch spiral CCTA constitutes an exception. p < 0.001). These results confirm those of a previous study using the same CCTA-planning approach . In our study, CCTA images were acquired using modern and low-dose prospective ECG-triggered scan protocols; consequently, we observed a median overall radiation dose of 3.2 [1.8–5.3] mSv. A study conducted by Leschka et al. on the same scanner observed an overall radiation dose of 9.0 ± 0.6 mSv for CCTA . The difOur study’s effective dose for the calcium-scoring scan was 0.33 mSv IQR, 0.25–0.37 mSv), which is slightly lower than in previous comparable studies . One exp–0.37 mSvOur study has several limitations. First, this was a retrospective, single-center, single-scanner trial. Therefore, the generalizability of the results is, by definition, limited. Second, all scout view CCTA plans were simulated, and a simulated scan length and radiation dose were calculated. In this respect, a prospective study design would have been more accurate and provided further value. Third, all the participants were included consecutively, and CCTA was clinically indicated; however, selection bias cannot be entirely excluded. Fourth, the analyzed prospective ECG-triggered scan protocols are vendor-specific. Lastly, due to the non-invasive nature of our study, we did not acquire invasive coronary angiography data and, therefore, were unable to assess the diagnostic accuracy of the proposed protocols against an invasive reference standard.However, our study is the first to investigate the role of CAS-CT as a planning tool for optimizing whole-examination radiation doses in low-dose modern prospective ECG-triggered CCTA. Prospective ECG-triggered CCTA acquisitions have replaced retrospective spiral scans in most clinical settings . Planning prospectively ECG-triggered CCTA via CAS-CT can potentially reduce the overall radiation dose of an examination compared to a scout view planning approach wherein no CAS-CT is acquired. A slightly opposite effect was only observed for high-pitch spiral CCTA.


Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease. The cholinergic synapse is among the most well understood synapses within many organisms, serving as a prototype for classical neurotransmission3. Amongst the primary molecular components of the cholinergic synapse are ACh binding receptors such as the ligand-gated nicotinic acetylcholine receptor channel (nAChR)4. In addition to their post-synaptic localization, nAChRs are also found presynaptically and can contribute to synaptic growth and neurotransmitter release in brain circuits for memory and cognitive processing5. The α7 nAChR is a widespread homopentameric channel receptor that activates calcium within cells6. Studies show that α7 nAChRs can signal through both ionotropic and metabotropic modes in neural and immune cells7. In particular, α7 nAChR signaling is important for neural cell development and synaptic growth10.Acetylcholine (ACh) is an abundant neurotransmitter in the brain and periphery important for various physiological functions including movement, memory, and immune system regulation11. AChE however is also a well-established signaling molecule with hydrolytic and non-hydrolytic functions including strong trophic activity14. The mammalian AChE gene contains six exons which are spliced in several alternative forms that create three main AChE isoforms 14. The synaptic tetrameric variant AChE-T is the dominant isoform in the brain14. AChE-T is a cell membrane attached enzyme via its well characterized proline-rich membrane anchor (PRiMA) domain14. AChE-T also has an amphiphilic region within its c-terminus that contributes to oligomerization15. Proteolytic cleavage of the last 30 amino acids at the c-terminus generates in vitro, as well as in vivo a T30 bioactive peptide17. Interestingly, since the c-terminal region of AChE-T contains some sequence homology with the amyloid precursor protein (APP), cleavage of AChE-T and APP appears to yield two peptides  with some sequence similarity , a powerful enzyme that regulates ACh levels within the synaptic cleft21. The pharmacological targeting of T30 is a promising approach for neurodegenerative and amyloid related disease22. In this study we explore how T30 impacts signaling within the human neural cholinergic cell line SH-SY5Y, which is a model for Alzheimer’s Disease (AD) and known to endogenously express ⍺7 nAChRs24. Using quantitative whole cell proteomics and targeted functional cell assays, we identify a novel mechanism of T30 signaling via mTOR that leads to cell growth. T30 activation of mTOR may provide a mechanistic understanding of how non-enzymatic AChE-T can contribute to synaptic function.Interestingly Aβ42 and T30 are reported to both bind to the ⍺7 nAChR, and can impact calcium signaling and neurotransmission2. Experiments were conducted in cells that did not exceed 19 passages. For treatment experiments, cells were grown to 70% confluence then treated with: 100 nM T30/T15/ NBP14, 50 nM α-bungarotoxin (bgtx) (Thermo Fisher B1601), 1 μM Rapamycin (Thermo Scientific AAJ62473MF). Treatment media was changed daily. T30, T15, and NBP14 peptides were provided by NeuroBio LTD  and have been characterized elsewhere26. Sequences for these peptides are listed in Supplemental Table 345–348A in pEYFP-C127 with the pEYFP-C1 plasmid used as a control. All cDNA constructs were propagated in DH5α cells (Thermo Fisher 18258012) and purified using a maxi prep kit . Cell proliferation was analyzed through live cell counting using phase contrast microscopy within a C-Chip hemocytometer .Human neuroblastoma cells SH-SY5Y cells (ATCC CRL-2266) were cultured in T75 flasks for propagation and then plated onto 100 μg/ml polyD-lysine  coated 96-well glass bottom plates for imaging. Cells were fed DMEM (Gibco 11995065) supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep at 37 °C and 5% COd-Lysine  and laminin  coated coverslips at 75,000 cells/well as previously described28. Cells were grown in a Neurobasal media with SM1 (StemCell cat# 05711), 12.5 μM glutamate, 500 μM glutamine, and 0.1 mg/mL Primocin (InvitroGen cat# ant-pm-1)] at 37 °C/5% CO2. At 2 DIV (days in vitro) cells were treated with 1 nM, 100 nM, 1 μM T30 or the vehicle (H2O) added to the culture media.For primary cultures, a total of 2 pregnant Sprague–Dawley rat mothers  were obtained from Charles River  with all animal experimental procedures approved in accordance with guidelines and regulations of the Georgetown University Institutional Animal Care and Use Committee and ARRIVE guidelines. All efforts were made to minimize distress during euthanasia. At embryonic day 19 (E19), pregnant rats were euthanized using a flow-regulated carbon dioxide chamber, and death was verified by toe pinch and decapitation. Anesthetics were not used prior to euthanasia due to interference of proper neuronal growth in hippocampal neuronal cultures. Primary hippocampal neurons were obtained from E18 Sprague–Dawley rats and cultured onto poly-29. In brief, at 3 DIV cells were lysed using a 0.1% Triton X-100 lysis buffer  supplemented with protease  and PhosSTOP (Sigma Aldrich 4906845001) inhibitors. Protein concentration was determined using the Bradford assay. Proteins were separated on a NuPAGE 4–12% Bis–Tris gradient gel (Thermo Fisher NP0322BOX) and then transferred onto a nitrocellulose membrane (Thermo Fisher IB301002). Membranes were blocked with milk prior to application of a primary antibody: GAPDH , LC3B , p-eIF4E , eIF4E (1:1000 Santa Cruz sc-271480), p-S6K1 (Thr389) , S6K1 (1:1000 Santa Cruz sc-8418) and Cytochrome C (1:1000 AbCam ab90529). HRP secondary antibodies were purchased from Jackson Immunoresearch . A SeeBlue Plus2 Ladder (Thermo Fisher LC5925) was used as molecular weight marker. Bands were visualized using SuperSignal West Pico or SuperSignal West Femto Chemiluminescent substrates (Thermo Fisher) via the G:BOX Imaging System and GeneSYS software . Band density was analyzed in Image J . All measures were normalized to GAPDH unless otherwise stated. Average band intensity measures are based on three separate experiments.Proteins were obtained from cultured cells as previously described30. Briefly, solubilized protein samples were incubated for 5 min with acetone on ice followed by protein precipitation via centrifugation. The resulting protein pellet was denatured, reduced, and alkylated with 8 M urea, 1 M dithiothreitol, and 0.5 M iodoacetamide. Proteins were digested with trypsin (0.5 μg/μl) in 500 nM ammonium bicarbonate and incubated at 37 °C for 5 h. The samples were then desalted with C-18 ZipTips (Millipore), dehydrated in a SpeedVac for 18 min and reconstituted in 0.1% formic acid before undergoing liquid-chromatography electrospray ionization mass spectrometry (LC-ESI MS/MS) with 5 technical replicates.Whole cell proteomic analysis was performed based on an established methodLC-ESI MS/MS was performed using an Exploris Orbitrap 480 equipped with an EASY-nLC 1200HPLC system . Peptides were separated using a reverse-phase PepMap RSLC 75 μm i.d by 15 cm long with a 2 μm particle size C18 LC column , and eluted with 80% acetonitrile and 0.1% formic acid at a flow rate of 300 nl/min. After a full scan at 60,000 resolving power from 300 to 1200 m/z, peptides were fragmented by high-energy collision dissociation (HCD) with a normalized collision energy of 28%. EASY-IC filters for monoisotopic precursor selection, internal mass calibration, and dynamic exclusions (20 s) were enabled. Data on peptide precursor ions with charge states from + 2 to + 4 was recorded.v2.4  was used to identify proteins by comparing raw MS peptide spectra to the NCBI 2018 human protein database using the following search engine parameters: mass tolerance for precursor ions = 2 ppm; mass tolerance for fragment ions = 0.05 Da; and cut-off value for the false discovery rate (FDR) in reporting peptide spectrum matches (PSM) to the database = 1%. Peptide abundance ratios were determined by precursor ion quantification in Proteome Discoverer v2.4, with the vehicle control group used as the denominator. Statistically significant abundance ratios with adjusted p-values < 0.05 were determined using a one-way analysis of variance (ANOVA) followed by Benjamini–Hochberg post-hoc analyses. Proteins with a quantifiable spectra signal profile seen in at least 3 of the 5 technical replicates were included in the analysis. Markov Cluster Algorithm (MCL) with an inflation parameter of 3 was used to perform clustering analysis on the data in the STRING database. Data was analyzed, organized, and presented using the R package : ggplot231, tidyverse32, Excel, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Search Tool for the Retrieval of Interacting Genes/Proteins  application33.The SEQUEST HT search engine within the Proteome Discoverer 8. α7 nAChRs were detected at the cell surface and within the cytoplasm using Alexa Fluor 488 conjugated α-bungarotoxin (Alexa-488 bgtx) (Thermo Fisher B13422) as described27. Images were captured using an inverted Zeiss LSM800 confocal microscope and the Zen software package .SH-SY5Y cells were fixed in a solution consisting of 1 × PEM  and 0.3% glutaraldehyde then quenched with sodium borohydride (2 mg/ml). Cells were permeabilized using 0.05% Triton X-100 (Sigma Aldrich). Quantification of structural change  was performed using rhodamine phalloidin (Cytoskeleton PHDG1-A). All morphometric measures were conducted and quantified using ImageJ  as describedhttps://imagej.net/software/fiji/downloads) via the Neuroanatomy plugin35.Primary culture coverslips were fixed at 7 DIV with 1% paraformaldehyde and 4% sucrose at room temperature for 7 min then submerged in methanol for 7 min at − 20 °C. Cells were immunolabeled with antibodies to MAP2  in a GDB solution consisting of 30 mM phosphate buffer (pH 7.4) containing 0.1% gelatin, 0.3% Triton X-100, and 450 mM NaCl at 4 °C overnight. Species specific secondary antibodies (AlexaFluor 488 (Invitrogen) were applied in GDB for 2 h at 25 °C, then washed, and coverslips mounted in NPG . Analysis of dendritic branching by Sholl analysis was performed on ImageJ v1.53t  analysis to define PPI networks within the T30 proteome 41. MCL analysis shows a relatively integrated PPI network based on the identity of the significantly altered proteins . This cluster (Cluster 1) consisted of an average local clustering coefficient (ALCC) of 0.697. Enrichment analysis of Cluster 1 confirms ribosome enrichment in KEGG Pathways with a false discovery rate (FDR) of 8.49 × 10–7. MCL analysis revealed 11 clusters within the T30-associated PPI network  networksins Fig. . Within 42. In particular, the c-terminal fragment T30 that is produced by AChE-T cleavage has been shown to activate intracellular signaling important for neural development44. Our proteomic analysis reveals new intracellular proteins and signaling pathways that are altered in response to a 3-day T30 presentation within the SH-SY5Y cell line. Bioinformatic KEGG Pathway analysis in DAVID further revealed enrichment of proteins involved in mTOR signaling. Our analysis indicates that mTOR may serve as a point of convergence between PPI networks and several of the identified clusters within the dataset . This effect was not seen in response to the application of a cyclic variant of the T30 peptide termed NBP14 that is shown to be biologically inert47  . As shown in Fig. 27. We first compared cell surface to intracellular expression by labeling cells with Alexa 488-bgtx in  non-permeabilized and permeabilized fixation protocols, respectively. Data shows that T30 increases the Alexa 488-bgtx signal within the cell  but not at the cell surface   that lacks the G protein-binding site has been established as a method for blocking α7 nAChR-mediated G protein signaling and diminishing the receptor's metabotropic effect on growth27. We tested the ability of T30 to promote growth in SH-SY5Y cells transfected with α7345–348A (pcDNA3-YFP). In this assay, control cells were transfected with the vector pcDNA3-YFP alone. Morphological analysis shows that T30 treatment does not increase neurite growth in cells expressing α7345–348A  unit α734–348A tha52. Previous work indicates that α7 nAChRs engage mTOR during development, inflammation, and cancer progression53. We directly confirmed the involvement of mTOR signaling in T30-mediated neurite growth using the mTOR inhibitor rapamycin. As shown in Fig. 55  protein undergo modifications during autophagy and thus serve as autophagy markers57. We examined autophagy-associated  LC3I to II conversion within LC3B as previously shown57. LC3B was detected throughout the cell, including sites of growth, consistent with the role of the autophagosome in modulating structural growth58 . In these experiments, the inactive portion of T30 (T15) did not have an effect on cytochrome C levels  ors Fig. A. A quan63. It is also widely expressed outside of the nervous system and is sometimes found in non-cholinergic cells64. A large body of work has demonstrated non-hydrolytic properties for AChE within various cell types14. Amongst its non-hydrolytic activity is its role as a trophic factor during normal development and cancer65. Studies show that synaptic AChE-T is especially abundant during brain development and can regulate axonal growth, cell adhesion, as well as pathfinding during synaptogenesis67. The loss of AChE-T in mice is associated with disruption to synaptic connectivity within the retina and cortex68. Our study supports the involvement of non-hydrolytic AChE-T in growth demonstrating an important role for the T30 peptide in human neural cells70. We add to this body of evidence by showing that T30 can signal growth in human SHY-SY5Y cells and primary hippocampal neurons. In neurons, we find that T30 activates dendritic growth within a specific concentration range, consistent with the idea that T30 allosteric ligand site within the ⍺7 nAChR35.AChE is an enzyme vital for mammalian synaptic transmission through its ability to hydrolyze ACh48. Sequence similarity between T30 and Aβ42 has been shown yet it is not yet clear if the two peptides share common binding partners in cells. Studies have shown that brain AChE complexes with Aβ peptide fragments71, and that AChE and butyrylcholinesterase are present within Aβ deposits such as senile plaque72. Interestingly, in ex vivo rat brain slices, the application of T30 results in an increase in the expression of Aβ4273, suggesting that T14 interferes with amyloid protein turnover. Interactions between AChE and nAChR have also been explored in other contexts74. Thus, while the two molecules are co-expressed at the mature cholinergic synapse, AChE-T and the ⍺7 nAChR appear highly coupled in expression during brain development75. In early post-natal synaptic development, α7 nAChR is at its highest within rodent brain and shown to regulate neural cell proliferation and synaptic maturation within regions such as the hippocampus10. Our earlier studies have shown an important role for α7 nAChR metabotropic signaling through G proteins in axonal calcium signaling and growth cone motility29. In this study, T30 activation of the α7 nAChR is also able to support neurite growth through a process that appears dependent on G protein signaling since expression of the α7345–348A mutant did not support  T30 mediated growth.Earlier findings demonstrate that T30 binds the ⍺7 nAChR77. It functions via two distinct complexes: mTORC1 and mTORC2 with the activation mTORC1 resulting in protein synthesis via p70 S6 kinase (S6K1 and S6K2) and phosphorylation of eukaryotic initiation factor 4-binding protein (4EBP1 and 4EBP2). mTORC1 also suppresses autophagy mediated protein degradation and can contribute to growth56. Our findings show that T30 promotes neural cell growth by activating α7 nAChRs leading to mTOR pathway signaling. This process is supported by recently published evidence on the ability of α7 nAChRs to direct AKT/mTOR autophagy within neurons79.Our proteomic analysis reveals several important intracellular pathways engaged by the presentation of T30 in vitro. These pathways all appear to promote cell growth and protein synthesis. In fact, when looking at statistically altered proteins within the T30 proteome, ~ 75% of the change was due to an increase in the expression of proteins. Bioinformatic analysis using MCL and DAVID KEGG pathway indicates that these protein changes reflect an mTOR pro-growth state within treated cells. mTOR is an evolutionarily conserved serine/threonine kinase that regulates many cellular responses (from autophagy to translation) and organizes molecular responses to incoming signals by modulating energy and metabolic substrates56. Interestingly, the effects of T30 appear accompanied by overall reduction in cellular cytochrome c, which is a driver of apoptosis80. The effects of T30 are found specific since antagonism of the α7 nAChR with bgtx abolished T30-associated growth, and application of the mTOR inhibitor rapamycin and non-bioactive peptide variants of T30 (T15 and NBP14) did not promote growth signaling. Studies have shown interactions between nAChRs and intracellular organelle including the ER and mitochondria19. In future studies, it will be important to examine subcellular compartment specific proteomic changes that accompany T30 activation of the nAChR.Our experiments show that T30 acts via mTOR at several points, first by decreasing the expression of the autophagy marker LC3B and second by increasing the phosphorylation of both eIF4E and S6K. These processes appear to promote neurite growth and explain the actions of T30 on growth within our cells as well as elsewhere50. In the brain stimulation of mTOR has been suggested to promote hyperphosphorylation of synaptic tau and can drive amyloid protein accumulation81. A growing body of evidence demonstrates a link between mTOR signaling and AD. For example, an alteration in the autophagy-lysosome pathway has been shown to drive Aβ42 neurotoxicity83, and a loss in mTORC1 regulation appears to contribute to protein aggregation within neural cells84. It has been suggested that interactions between T30 and nAChRs can participate in early cholinergic cell death within the brain85. This study provides novel evidence on a connection between the mTOR pathway and ⍺7 nAChR signaling. How this may contribute to neural development and disease is an important question for the future studies.Disruption to mTOR signaling is implicated in many human disease including auto-immune disorder, neurodegeneration, and various cancersSupplementary Information.


One of the routine clinical treatments to eliminate ischemic stroke thrombi is injecting a biochemical product into the patient’s bloodstream, which breaks down the thrombi’s fibrin fibers: intravenous or intravascular thrombolysis. However, this procedure is not without risk for the patient; the worst circumstances can cause a brain hemorrhage or embolism that can be fatal. Improvement in patient management drastically reduced these risks, and patients who benefited from thrombolysis soon after the onset of the stroke have a significantly better 3-month prognosis, but treatment success is highly variable. The causes of this variability remain unclear, and it is likely that some fundamental aspects still require thorough investigations. For that reason, we conducted in vitro flow-driven fibrinolysis experiments to study pure fibrin thrombi breakdown in controlled conditions and observed that the lysis front evolved non-linearly in time. To understand these results, we developed an analytical 1D lysis model in which the thrombus is considered a porous medium. The lytic cascade is reduced to a second-order reaction involving fibrin and a surrogate pro-fibrinolytic agent. The model was able to reproduce the observed lysis evolution under the assumptions of constant fluid velocity and lysis occurring only at the front. For adding complexity, such as clot heterogeneity or complex flow conditions, we propose a 3-dimensional mesoscopic numerical model of blood flow and fibrinolysis, which validates the analytical model’s results. Such a numerical model could help us better understand the spatial evolution of the thrombi breakdown, extract the most relevant physiological parameters to lysis efficiency, and possibly explain the failure of the clinical treatment. These findings suggest that even though real-world fibrinolysis is a complex biological process, a simplified model can recover the main features of lysis evolution. It occurs when an aggregate of blood components clogs a brain artery, thus impeding sufficient irrigation. These thrombi, or blood clots, are mainly composed of red blood cells (RBCs) and platelets bound together by fibrin fibers, although they may contain several other biological entities. The current treatment options are either thrombolysis alone, a biochemical breakdown of the thrombus, or used with a mechanical removal of the clot . The latter, whose efficacy has been demonstrated4 requires endovascular treatment and is indicated only in large, easily accessible arteries. The former, less invasive and applicable in first aid within a few hours after stroke symptoms onset, is done by injecting a pro-fibrinolytic macromolecule into the bloodstream. This agent, typically tissue-Plasminogen-Activator (tPA), binds to the fibrin fibers to facilitate the conversion of plasminogen to plasmin, which will then cut the fibrin strands and result in thrombus breakdown. Although the biochemistry of fibrinolysis is well understood5, our knowledge of many aspects of the complete process in vivo and in vitro remains sparse7. For example, the permeability of the thrombus, which is central to the transport of active macromolecules in the bloodstream, is only estimated via indirect measurements like perviousness8; the exact amount of drug that reaches the thrombus is unknown; even the dynamics of fibrin strands degradation is still actively investigated14. Even though patients who receive early intervention show a better three-month prognosis, about fifty percent fail to respond, and the reasons for this are unknown. Hence, theoretical, experimental, and clinical research are essential to understand the underlying mechanisms of the variability of treatment outcomes and eventually improve further drug treatment development.Ischemic stroke, already reported by Hippocrates in 400 B.C.We present in this study a combination of experiments and modeling to investigate the dynamics of flow-driven lysis profiles of fibrin-pure clots. Experimentally, we create in a tube in vitro an occlusive fibrin clot in a tube, mimicking a thrombus blocking a blood vessel. We monitor the progress of the lysis and estimate the front lysis profile’s time evolution and the flow’s dynamics seeping through the clot driven by inlet and outlet pressure difference. We monitor the progress of the lysis and estimate the time evolution of the lysis profile and the dynamics of the flow seeping through the clot. The experiments show that the lysis accelerates in time. However, the flow remains constant or slightly increases during the lysis, thus suggesting that the recanalization would occur late. We translate these observations into a simple one-dimensional lysis model with constant flow and all pro-fibrinolytic agents aggregated into a single species, called anti-Fibrin Agent (anti-FA), that is blocked at the front of the clot and interacts through a second-order reaction. This model can be solved analytically and accurately predicts lysis profiles given the initial conditions.15 and combined compartmental models for the temporal evolution of the proteins concentration, convection-diffusion-reaction equations to describe the transport of their free phase, and the reactions of bound proteins. Convective transport, due to blood flow, is a central issue and mainly relies on the Navier–Stokes equations, considering the clot as a homogeneous medium17 or the blood as a homogeneous visco-elastic fluid15, or relying on Darcy’s law with constant pressure drop18. Some models use either a small pressure drop16, a high concentration of the profibrinolytic proteins19, or accelerated kinetics20. Nevertheless, they provide valuable information on simulated thrombolytic treatments, the extent of lysis, and changes in flowrate and pressure but are not directly compared to experiments realized in similar conditions.Several numerical models have already tackled the flow-driven thrombolysis question. Most of them were inspired by Anand et al.’s seminal work21. These authors previously concluded that deterministic models of fibrinolysis might be inappropriate given the concentrations involved22, but the multiscale model considers only diffusional transport. A mechanistic microscale model, where the clot is modeled as red blood cells trapped in a regular assembly of elastic fibrin bonds, described the removal of clot fragments due to biochemical and mechanical degradations23. This fine-grained model investigates the role of flow conditions on clot fragmentation but resolves thrombolysis up to a second of physical time.Another approach considers multiscale fibrinolysis with microscale detailed stochastic biochemistry and lysis of a single fiber and macroscale degradation of the full clot24 instead of solving the Navier–Stokes equations. The fibrin clot is described as a porous material, with porosity changing locally with the progress of the lysis. Our framework can incorporate heterogeneity directly into clot porosity and composition to assess to what extent the thrombus heterogeneity can impact stroke treatment and determine whether it is possible to design novel protocols when considering thrombolysis in acute ischemic stroke.In this study, we rely on a mesoscopic approach in which the flow is computed with the open-source lattice-Boltzmann (LB) library PalabosPasteur pipettes, with the tips cut off, were used (VWR). To form the fibrin clot at a height suitable for the experiments, we placed 900 The Pasteur pipettes, with clot inside, are then placed on a stand and a solution is added to a height of 2 cm. This consists of Owren buffer, tPA (Merck) at a final concentration of 10 A variant of the experiment without plasminogen was performed to measure the migration of tPA. Alone the tPA is added only in the supernatant. The clot is collected after a migration time of 30, 60, and 120 min. The clot is then cut into four equal parts. Plasminogen is added and the whole is incubated for 16 h. The lysate is then collected for tPA assay by Elisa (Invitrogen). In another experiment, the clot is allowed to lysate over the first quarter of its length. The clot is then removed and cut into four equal parts, left to incubate for 16 h and the lysate is recovered for tPA determination by Elisa (Invitrogen).25, and the progression of the lysis was evaluated by measuring the distance between the initial clot position and the interface between the clot and the solution. When the interface was not horizontal, especially at the end of the lysis, the front position was measured at the middle of the interface. To evaluate the flow, we collected the liquid during successive 10-min intervals and measured the corresponding volume that passed through the clot.To monitor the evolution of the lysis, pictures of the tubes were taken every 10 min, with the initial position of the clot marked on the tube. Each image was manually analyzed using the software ImageJ21. In17, the authors have shown that the number of equations necessary to describe the lysis of fibrin pure clot can be reduced to only 6. Hence, we wonder whether it is possible to lower this number even more, while preserving the main features of the lysis. To do so, we propose a one-dimensional minimal model of lysis that relies on few assumptions and that can be solved analytically. This model aims not to replace previous detailed models of fibrinolysis but rather to identify a set of minimal rules sufficient to reproduce the time evolution of the experimentally observed lysis profiles. We consider that the system is made of only two species. The first one is the fibrin (F), and the second, the anti-FA  and L and fibrin concentration n slices L, this result is independent of Typical models of fibrinolysis consist of ten or more equations to account for the different reactions between species, free or bound, involved in the fibrinolytic cascadeWe have demonstrated that considering only one profibrinolytic species blocked at the front of the clot is sufficient to recover non-trivial lysis profiles. We now incorporate those findings in a more elaborated three-dimensional model that accounts for the fluid simulation, the thrombus as a porous medium, and the lysis. These elements are summarized in Fig. 27, the LB method is an efficient alternative to the NS equations32, that has been widely used to address biomedical problems35. Briefly, it solves the Boltzmann equation, which describes the evolution of so-called “fluid” particles in a regularly discretized lattice. The spatial discretization enforces a set of q discrete velocities 36:37):31). Finally, the streaming step propagates the scattered populations:In order to simulate the fluid flow, we rely on the so-called lattice-Boltzmann (LB) method, which allows the simulation of fluids without explicitly solving the Navier–Stokes (NS) equations. The LB method can be seen as a latticed discretization of the kinetic theory, where the probability density function is replaced by fictitious particles colliding and streaming on the lattice. Inspired by Lattice Gas Automatamentclass2pt{minim18, and the thrombi are typically millimetric to centimetric38. Simulating the lysis of such an intermingled mesh at the fiber scale seems unrealistic and computationally expensive, considering the size and time scales involved. We aim to simulate the lysis of centimetric clots over hundreds of minutes while state-of-the-art detailed molecular models of fibers operate at nanometer and microsecond39. Instead, with a coarser description, one can treat clots as fibrous porous media41, which can be done in the context of LB42 with the so-called partial bounce-back (PBB) method.Fibrinogen is a soluble blood protein made up of three pairs of polypeptide chain, and converted into fibrin by the cleavage of the fibrinopeptide by thrombin. Due to a change in solubility, fibrin monomers polymerize into protofibrils that aggregate into fibers that merge to form a branched network of fibers. The resulting fibrin fibers’ diameter range from b denotes a point at the boundary and i. The PBB method is an extension of the solid bounce-back boundary. The underlying idea is to assign a continuous fraction of bounce-back to each volume element, whether none, part, or all of the fluid is reflected at the boundary , while mentclasspt{minima26, the authors derived analytically the relationship between the permeability k and the bounce-back fraction:k. It implies that in order to simulate a porous material with the characteristic permeability of a thrombus, we have to incorporate in our model a permeability law that provides a value of k, knowing the radius of the fibers  varies non-linearly with time; consequently, the lysis velocity increases as the clot is lysed. Previous numerical models found different lysis profiles, typically linear or sub-linear17. This difference can be explained by the fact that simulated bound tPA at the front of the clot tends to decrease with time, while our experimental data indicate a constant accumulation at the front . 3D reconstructed thrombi could also be lysed, provided 3D data is available. We chose in the present work to treat heterogeneity as a change in fibrin concentration, but other options could be to change locally the reaction rate The spatial thrombus heterogeneity alone showed a significant influence on lysis time and recanalization, with up to 59 identified up to 4 different patterns of flow in clotted regions of ischemic stroke patients: slow anterograde, fast anterograde, retrograde and mixed anterograde–retrograde flows. Anterograde flow designates the expected direction of the flow, i.e. from proximal to distal, while retrograde means the opposite direction. These different flow patterns undoubtedly affect the transport of lytic drugs to the occlusion site and, eventually, the evolution of the lysis. Also, the artery is here modeled by a rigid tube, and clot fragments stay in place until totally lysed, even when disconnected from the wall or the main clot chunk. Fortunately, thanks to its design, the model could be easily extended to include conditions closer to in vivo thrombolysis. We could instead reconstruct60 patient-specific geometries with collateral vessels and try to reproduce the complex flow conditions observed in AIS patients59. In doing so, we could hypothesize the effect of artery geometry, thrombus location, and drug injection patterns on lysis efficacy. Seners et al. have reported that distal thrombi lyse faster than proximal thrombi61. One could use the numerical model to assess whether this discrepancy comes from the shape and composition of distal clots, local flow conditions, or both. The model may estimate the risk of thromboembolism by accounting for the interaction of the disconnected clot components with the fluid. Using methods for moving boundaries or modifications of the PBB approach63, would allow to make these components move with the fluid, aggregate with other fragments, re-adhere to the main clot, or eventually leave the system, hence mimicking thromboemboli. Future works will incorporate these processes to extend the model to assess whether certain conditions in our numerical experiments are more likely than others to form emboli.The numerical model is purposely streamlined, keeping only the essential features needed to replicate the experimental observations. In that regard, it suffers in appearance from not seeming clinically relevant. For instance, we assume steady and laminar flow, whereas it is not the case in vivo. Recent work from INSIST partnersDuring the last two decades, we have witnessed an unprecedented revolution in treating acute ischemic stroke. Indeed, intravenous thrombolysis and endovascular mechanical thrombectomy therapies have entirely changed patient outcomes. However, disappointingly, while the endovascular mechanical thrombectomy allows for up to 85% of reperfusion in acute ischemia related to large vessel occlusion, the intravenous thrombolysis is still associated with low recanalization rates, less than 30%, in large and medium-sized vessels. Understanding conditions that increase lysis rates is essential to improve stroke therapies and should help explain clinical failures. In summary, we proposed a versatile and evolutive framework combining modeling and in vitro experiments to improve our understanding of fibrinolysis of pure fibrin occlusive clots in a tube. The one-dimensional model is purposely simplified to be solved analytically to predict front lysis time evolution. In future work, we aim to extend the model to incorporate more details of the clot structure and investigate further lysis patterns, including fragmentation, of clots with heterogeneous permeability and compositions. We intend to provide valuable knowledge to understand better the thrombus’s pathophysiology and its treatment with intravenous thrombolysis to improve the patient’s care.Supplementary Information.


There are concerns that antimicrobial usage (AMU) is driving an increase in multi-drug resistant (MDR) bacteria so treatment of microbial infections is becoming harder in humans and animals. The aim of this study was to evaluate factors, including usage, that affect antimicrobial resistance (AMR) on farm over time.A population of 14 cattle, sheep and pig farms within a defined area of England were sampled three times over a year to collect data on AMR in faecal Enterobacterales flora; AMU; and husbandry or management practices. Ten pooled samples were collected at each visit, with each comprising of 10 pinches of fresh faeces. Up to 14 isolates per visit were whole genome sequenced to determine presence of AMR genes.Sheep farms had very low AMU in comparison to the other species and very few sheep isolates were genotypically resistant at any time point. AMR genes were detected persistently across pig farms at all visits, even on farms with low AMU, whereas AMR bacteria was consistently lower on cattle farms than pigs, even for those with comparably high AMU. MDR bacteria was also more commonly detected on pig farms than any other livestock species.The results may be explained by a complex combination of factors on pig farms including historic AMU; co-selection of AMR bacteria; variation in amounts of antimicrobials used between visits; potential persistence in environmental reservoirs of AMR bacteria; or importation of pigs with AMR microbiota from supplying farms. Pig farms may also be at increased risk of AMR due to the greater use of oral routes of group antimicrobial treatment, which were less targeted than cattle treatments; the latter mostly administered to individual animals. Also, farms which exhibited either increasing or decreasing trends of AMR across the study did not have corresponding trends in their AMU. Therefore, our results suggest that factors other than AMU on individual farms are important for persistence of AMR bacteria on farms, which may be operating at the farm and livestock species level. The increasing prevalence of antimicrobial resistance (AMR) is currently one of the greatest threats to global public health . AMR incCampylobacter in Canadian sheep farms found that most AMU-AMR associations were not statistically significant  and feed practices were less commonly detected ] within a defined geographical area . All farms within the area were identified, and those with the largest herd sizes invited to join the study, to ensure commercial-sized farms were recruited. Five cattle and sheep farms provided consent to join the study  although only four pig farms could be recruited due to the lack of available pig farms willing to participate, leaving a population of 14 farms . Full deEach farm was visited on three seasonal occasions in 2017. Winter visits occurred between January and April, summer visits between June and July and autumn visits October to November. Individual visits to each farm were planned to allow at least 3 months between them.Each farm was split into up to five epidemiological groups, representing animals managed in the same way . At each visit, 10 pooled fecal samples were collected, with each pooled sample made up of 10 small pinches of fresh feces collected from the floor using a gloved hand and combined to form a golf ball-sized sample. The 10 pooled samples were then pooled again to create a single composite sample per farm. The sampling locations were chosen to be representative of the farm, with roughly two samples per epidemiological group, although this was adjusted where less than five groups were present or a disproportionate number of animals occurred in each group. When collecting samples from each group the samples were taken from different locations to be representative, e.g., in a building of 20 pens, every second pen had a pinch taken from it, or fields were split into sectors and pinches taken from each sector.At each visit a group-level record was completed, characterizing weather conditions at the time of sampling, the number and type of animals present in each group and details of their housing types or pasture locations. Additionally, a farm-level questionnaire was completed by farmers directly before the first sampling visit to capture farm structure and management information, such as hygiene, disinfection, and biosecurity practices. Any relevant changes to the questionnaire topics were noted by sampling staff at subsequent farm visits. AMU in the preceding 3 months was also documented for all farms, with details collected on products used and type and number of animals treated. Additionally, two pig farms gave consent for access to their electronic Medicine Book (eMB) , recordiEscherichia spp., Citrobacter spp. and Klebsiella spp.Faecal samples were diluted in buffered peptone water, plated on to CHROMagar™ ECC  and 14 colonies were selected. MALDI-TOF  or 16S rRNA sequencing  was usedCitrobacter spp. and Klebsiella spp. were excluded , as well as sequence data assembled for further analysis as described in excluded .Questionnaire data were cleaned prior to analysis, with missing values replaced by values of “not known” or “not applicable.” The total weight of each active antimicrobial ingredient (mg) was calculated, to allow the determination of milligrams per kilograms (mg/kg) of animals treated. European standardized animal weights were used to determine the estimated average kg weight of each animal type named as being treated by each product at time of treatment . These cThe AMU data and the genotype results for each antimicrobial class were summarized per farm and per visit. Isolates were classified as MDR, if they had genotypic resistance to more than two antimicrobial classes . The AMRp < 0.05) associated with an isolate being MDR. The unique farm identifiers (recorded as RH01-RH15) were included in the model as a random effect to account for farm-level clustering. An initial univariable screening stage was used to assess all independent variables, with those with a p-value>0.25 omitted from further analysis. A backwards stepwise method was then used to select variables to be retained in the model. At each step variables with the greatest p-value were removed from the model and a Likelihood Ratio test was used to determine whether the removal significantly improved the fit of the model. The final multivariable model was fitted from variables retained in the model that had a Likelihood Ratio p-value<0.05. Where variables were collinear , after univariable assessment, only the best fitting variable was progressed to the multivariable step.The farm and group level data collected at each visit were assessed to detect any changes in management and these ‘change’ variables were combined with the AMU data and AMR results and imported into STATA 16  for analysis. A mixed-effects logistic model was used to identify significant risk factors  or E. fergusonii  but other Escherichia spp.  were identified as well as Citrobacter spp.  and Klebsiella spp.   . AMR genEscherichia, Citrobacter, and Klebsiella isolates had relatively similar AMR genotypes, whereas the single isolates of Escherichia marmotae and Klebsiella oxytoca were dissimilar . The mean number of AMR classes, in which genes conferring resistance to a particular AMR class were detected, differed between animal species . More weE. coli, 12 (23.5%) E. fergusonii, 7 (22.6%) Citrobacter spp., and 3 (27.3%) Klebsiella spp. We observed a wide diversity of AMR profiles at the class level in the 129 genotypically resistant isolates, with 8, 26 and 3 different profiles in isolates from cattle, pigs, and sheep, respectively.The most common MDR profile combined aminoglycoside, beta-lactam, trimethoprim, sulphonamide and tetracycline resistance genes. This was also the most common AMR profile in isolates from cattle and sheep farms, whereas the most common MDR isolates in pigs also included additional amphenicol resistance genes . MDR wasAMR genes were infrequently detected in isolates from visits to the sheep farms . For catThe standard deviations of the proportion of isolates with AMR were consistently greater than the mean for cattle isolates for all antimicrobial classes , indicating larger variation in the proportion of resistant isolates per farm visit . The summphAB/ mefB) (17% from RH01 and 58% from RH02). Quinolones are listed as a HP-CIA, and macrolides are an WHO HP-CIA, although not classed as such by the European Medicines Agency. These genes were harbored in addition to commonly detected AMR genes, such as those conferring sulphonamide and tetracycline resistance. Also, a significantly higher proportion of E. coli from RH02  harbored PMQR plasmids or MLS  resistance compared to E. coli from RH01 .Multi-dimensional scaling summarizing the isolate AMR genotype profiles by farm showed a tight cluster of farms  with few AMR isolates (range 0–3) and only 0–1 MDR isolate per visit . Pig farThe most commonly used antimicrobial classes were the non-extended spectrum cephalosporins (non-ESC), with usage reported in the 3 months prior to 26/42 (62%) farm visits, and aminoglycosides, with use reported prior to 22/42 (52%) visits. Total AMU over three 3-month periods preceding all three visits indicated that pigs and cattle on average used the most amount (in weight) of different antibiotic products, active ingredients, and antimicrobial classes in total per farm, when compared to sheep . The totGenerally, the number of antimicrobial classes used was relatively consistent across the three visits for each farm, varying by only one added or removed class across visits for four of the cattle farms (RH06 and RH08-RH10), three sheep farms (RH13-RH15) and three pig farms (RH02-RH04) . A furthWhen the AMU was compared to AMR for each farm over the three visits, decreasing or increasing resistance trends to at least one antimicrobial class were visualized for five farms: three pig and two cattle farms. These farms were assessed to explore the patterns of AMU and AMR for specific antimicrobial classes ; howeverInterestingly, three farms had AMR isolates detected at only a single visit but had reported AMU throughout the study . Cattle p < 0.25), and 6 were significant (p < 0.05) at this stage, only a single variable was retained in the final multivariable risk factor model, which was the number of unique antibiotic classes used in the 3 months before a visit (R2). For each additional antibiotic class used, the risk of an isolate being MDR increased by 1.6 (p = 0.002).The risk factor model for MDR outcomes assessed the presence of significant association of MDR bacteria with 12 husbandry/ management or seasonal conditions variables and 12 AMU variables . Although 14 variables progressed from the univariable screening stage  did not have the same degree of AMR as the two high usage pig farms, whereas the pig farms with low AMU had moderate proportions of AMR detected. These results therefore suggest that factors other than AMU alone are influencing AMR on farms, and that these may be operating at the farm and livestock species level.Significant linear associations were not detected between AMU and on-farm AMR by antimicrobial class, at the isolate level. This may relate to differences in the mechanisms of resistance for specific antimicrobials within each class, e.g., apramycin use may not have correlated with aminoglycoside resistance. The investigations of those farms which exhibited either increasing or decreasing trends of AMR across their visits did not have corresponding trends in their AMU. Additionally, a cattle farm (RH09) which presented relatively high AMU . The difference in AMU by livestock species is supported by AMU monitoring in the year of this study (2017) across the UK, with only 3% of active ingredients from pig farms being injectables compared to 69% on cattle farms  can be found at: Ethical review and approval was not required for the animal study because Samples were only collected from the environment of the animals and not direct from the animals. Ethical approval was not sought as sampling from faecal deposits on the ground was deemed outside of the Animal (Scientific Procedures) Act 1986.RS, MA, NS, and DR: study design. HM and DG: data collection. MAO, ES, LS, and KC: laboratory sample analysis. RS, HM, MAO, DG, and MV: data analysis. RS, MV, HM, MAO, and MA: manuscript writing. DC and MA: review of study. All authors: review of manuscript. All authors contributed to the article and approved the submitted version.This work was funded by the Antimicrobial Resistance Cross-council Initiative supported by the seven research councils [NE/N019989/1 and NE/N019660/1], and supported by National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at the University of Oxford (NIHR200915) in partnership with Public Health England (PHE). Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute, supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. The publishing of the manuscript was funded by the Veterinary Medicines Directorate under project VM0533. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England. This work was supported by the NIHR Oxford Biomedical Research Centre. KCC is Medical Research Foundation-funded. DWC, TEAP and ASW are NIHR Senior Investigators. The funders had no role in the design of the study, analyses, interpretation of the data or writing of the manuscript.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.


Ocular pyogenic granuloma is a benign vascular tumor that occurs primarily in children. Treatment options for pyogenic conjunctival granulomas include topical steroids, topical timolol, surgery, cryotherapy, and electrocautery. Patients with giant pyogenic granulomas are usually treated with surgical intervention. In this case, a 13-year-old Egyptian girl developed a giant pyogenic granuloma after strabismus surgery. Topical steroids showed a poor response and failed to demonstrate any improvement. While on timolol, the granuloma completely regressed, with no signs of recurrence. Despite the usual surgical approach to the treatment of purulent giant granulomas, we believe that topical timolol can be the preferred option as a noninvasive alternative therapy since it is considered safe when compared to the potential risks of topical steroid therapy or surgical exposure. Ocular pyogenic granuloma (PG), also known as lobular capillary hemangioma, is a benign vascular tumor that occurs mainly in younger people and children . There aThese lesions often develop on cutaneous or mucosal surfaces . The mosThey are diagnosed in the clinical setting and confirmed histologically. They usually bleed or cause irritation or discomfort, prompting patients to seek treatment . TreatmeWe report a case of giant conjunctival PG after strabismus surgery that completely regressed after topical treatment with timolol 0.5%. No adverse effects were reported.This article was previously presented as a meeting abstract at the 7th Health Professions Conference on December 25, 2022.A 13-year-old Egyptian girl, medically free with the exception of strabismus, underwent uneventful strabismus surgery with bilateral medial rectus recession using polyglactin 910 (Vicryl) for partial accommodative esotropia.Two weeks after surgery, the patient presented with mild redness at the surgical sites in both eyes. No history of trauma, itching, discharge, or rubbing was documented.One month after surgery, the patient presented with a giant conjunctival granuloma on the left eye. It was a red, congested, bloody, mushroom-like protrusion with an estimated base of 8 mm, height of 5 mm, and width of 5 mm, located in the nasal area of the left bulbar conjunctiva, and exhibiting a progressive pattern Figure .Eye examination revealed orthotropia with the prescribed glasses. Slit-lamp examination of both eyes showed a clear cornea with a deep anterior chamber.Post-strabismus surgery, the patient received 1% prednisolone acetate drops for the left eye every eight hours for two weeks, then the dosage was increased to two drops per hour, and then, she was switched to loteprednol etabonate 0.5% for two weeks once daily. No improvement was noted Figure .Because of the constant progression of the granuloma, timolol maleate 0.5% was introduced to the left eye every 12 hours. After the introduction of timolol, the patient showed remarkable improvement, and the topical steroid was slowly discontinued Figure .Complete regression was observed after three months following the surgery Figure .Follow-up examinations were performed regularly at three months, six months, and 18 months; there was no evidence of recurrence Figure .The exact mechanism that causes pyogenic granulomas (lobular capillary hemangiomas) to develop is unknown. An imbalance in angiogenesis regulation is proposed as a mechanism involved in the formation of pyogenic granulomas . To a ceFurthermore, many factors, such as trauma, chalazion, or after ocular surgery, are believed to have a causal association with pyogenic granuloma . A studyA pyogenic granuloma begins as a small, reddish papule, which progresses over weeks to months in size but does not grow beyond 1 cm ,12. MostBy this, the diagnosis of pyogenic granuloma is straightforward, depending on the clinical presentation. It commonly presents externally at the palpebral conjunctiva, along with a history of a dome-shaped papule or erythematous with or without bleeding over a couple of days or weeks -15.Further histopathologic examination is essential to exclude conditions that mimic pyogenic granuloma. Using hematoxylin and eosin (H&E) stain on the obtained section shows the formation of lobulated capillaries with fibrous septation and non-granulomatous inflammatory cells surrounded by edema ,15.Even though some individuals may have spontaneous healing, pyogenic granulomas typically need treatment due to recurrent ulceration and bleeding. Treating pyogenic granulomas can be tricky, as patients respond differently to a variety of treatment modalities. The aim of the treatment is to prevent further growth, recurrence, and any potential bleeding that might occur . FurtherThere is no unanimity or agreement on the optimal management of pyogenic granuloma, but different approaches are practiced and used for its management. The selection of the treatment technique depends on different factors, including size, location, patient age, and risk of recurrence ,12.Treatment modalities have been prescribed in a retrospective study. A total of 19 different treatment regimens were used on 1162 cases of PGs. The most common method used is surgical excision, which consists of either shaved excision or curettage followed by laser therapy or full-thickness excision .Complete excision is preferred in terms of preventing recurrence and obtaining a specimen for histopathological examination . SurgicaSclerosing agents, such as polidocanol, are also being used as a modality for the treatment of pyogenic granulomas . Non-surThe size of a granuloma can be efficiently curtailed with topical corticosteroid treatment. To anticipate the probable adverse effect, it is necessary to keep a careful eye on the intraocular pressure and taper the dose. If the lesion does not respond to corticosteroids, surgical excision and histological examination are suggested ,24. Topical timolol has been shown to be effective in the treatment of conjunctival pyogenic granuloma in a modest number of children and adults without major side effects . There hTopical timolol has been used safely; however, a significant reversible reduction in intraocular pressure was reported using timolol, which resolved after the treatment course . In addiTable In the present case, despite the usual surgical approach to giant pyogenic granuloma management, we believe that topical timolol can be the preferred option as a non-invasive alternative therapy for pyogenic granuloma with consistent follow-up and a good assessment of the case.This approach is very safe in comparison to the possible risks of topical steroid therapy or surgical exposure, especially with general anesthesia.