Nearly 70 years old dream of incorporating molecule as the device element is still challenged by competing defects in almost every experimentally tested molecular device approach. This paper focuses on the magnetic tunnel junction (MTJ) based molecular spintronics device (MTJMSD) method. An MTJMSD utilizes a tunnel barrier to ensure a robust and mass-producible physical gap between two ferromagnetic electrodes. MTJMSD approach may benefit from MTJ's industrial practices; however, the MTJMSD approach still needs to overcome additional challenges arising from the inclusion of magnetic molecules in conjunction with competing defects. Molecular device channels are covalently bonded between two ferromagnets across the insulating barrier. An insulating barrier may possess a variety of potential defects arising during the fabrication or operational phase. This paper describes an experimental and theoretical study of molecular coupling between ferromagnets in the presence of the competing coupling via an insulating tunnel barrier. We discuss the experimental observations of hillocks and pinhole-type defects producing inter-layer coupling that compete with molecular device elements. We performed theoretical simulations to encompass a wide range of competition between molecules and defects. Monte Carlo Simulation (MCS) was used for investigating the defect-induced inter-layer coupling on MTJMSD. Our research may help understand and design molecular spintronics devices utilizing various insulating spacers such as aluminum oxide (AlOx) and magnesium oxide (MgO) on a wide range of metal electrodes. This paper intends to provide practical insights for researchers intending to investigate the molecular device properties via the MTJMSD approach and do not have a background in magnetic tunnel junction fabrication. From 1950 to 1990, silicon technology grew exponentially while the lack of tools and technology forced molecular device research in its nascent state. From the early 1990, as attempts for making molecular devices gained momentum, the spintronics research grew tremendously and became commercially successful2. The parallel growth of two fields led to a new field of molecular spintronics3. Molecular spintronics devices (MSD) are promising for giving performance better than conventional silicon and spintronics devices4 and a candidate for providing hardware for quantum computers6. MSD based spin valves utilizing the electron's spin can be formed by chemically stitching molecular channels to the source and drains made up of magnetic materials or ferromagnets (FMs)8. However, it is a daunting task to maintain the molecule dimension (~ 0.5–3 nm) robust and reproducible gap between the two magnetic thin film layers11. Conventional molecular device fabrication approaches largely went backstage as they were not able to yield robust and mass-producible MSD based computer devices11. Specifically, the primitive MSD fabrication approach required placing the molecular monolayer between the electrodes3. However, molecular monolayer(s) are not dense and can be punctured due to inter-electrode metal diffusion and structural defects within molecules themselves11. Resultantly, defects within molecular monolayers create a direct exchange coupling that can compete or overturn the coupling via molecules and eventually may create a short circuit3.Quest for utilizing molecule as a computer device element begun along with the mainstream silicon devices around seven decades before12. However, the atoms that migrated during electromigration to create a nanogap may persist in the molecule's vicinity and create a competing coupling between the electrodes13. Additionally, mass production is extremely challenging due to the irreproducible break-junction profiles generated after electromigration or mechanical breaking of a nanowire.Notably, most of the experimental molecular spintronics research has focused on nanogap devices. Such molecular devices are based on creating a break-junction in a nanowire to produce molecule length scale gap by electromigration2, we have explored a magnetic tunnel junction (MTJ) based MSD . This MTJMSD method is intended to overcome limitations associated with conventional approaches. The MTJMSD is an excellent testbed for understanding and controlling the impact of coupling between the electrodes via defects in a molecular device14. This approach, utilizing commercially successful MTJ technology2  methods to create a general understanding.This paper extensively discusses the example of the AlOx tunnel barrier-based MTJMSDs and elaborates a diagnostic method to identify the evolution of defects using a ferromagnetic resonance study. In the case of AlOx, we observed that several hillock-like features appear over a period after the MTJ testbed fabrication Fig. c. The heJi) in the presence of molecule-induced exchange coupling with ferromagnets  We did not pursue DFT to explain and understand near room temperature behavior of MTJMSD. Conventionally used DFT approaches fundamentally work for absolute zero temperature. DFT approach is unable to simulate the effect of a wide range of thermal energy on an MTJMSD. Studying the role of thermal energy is critical because magnetic materials and magnetic molecules change dramatically with temperature30. (iii) Also, DFT is exceptionally challenging, if not impossible, to simulate the role of a wide range of defects in a system comprising thousands of transition metal atoms in a magnetic tunnel junction and molecular magnets. By varying the defect-associated parameter in MCS, Ji in Fig. Ji is consistent with the prior theoretical simulation on MTJ. In the prior work, defects within the tunnel barrier impacted the exchange coupling (akin to Ji in this paper) between two FM electrodes of an MTJ25. (v) We were unable to find a simulation method that could simulate the role of defects for the case of molecule-induced strong exchange coupling as experimentally observed in our prior work31. By parametrically varying molecular coupling factors  Our prior MCS study provided insights about the intriguing experimental observations on MTJMSDs for the cases where paramagnetic molecules produced strong exchange coupling18. The inter-FM electrode magnetic coupling occurs via the molecules and via the insulating layer between two FM electrodes  and right FM (JmR) electrodes.To practically accomplish the MCS study with desktop workstations in a reasonable time, the size of the MTJMSD Heisenberg model was fixed within mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minim35. The continuous model allowed spin vectors to settle in any randomly selected direction in a spherical coordinate system and hence have 3D vector components according to the equilibrium energy governed by Eq.  competing with molecules. The growth mechanism of the ultra-thin insulator on various types of the substrate may lead to non-uniform thickness across the planar area. Also, interfacial mismatch strains may lead to point defects24 becoming a source of Ji. Additionally, the residual tensile stress during fabrication, annealing22, and charge transport36 may cause the tunnel barrier fragmentation, producing pinholes-like features. Such defects can become the basis of direct exchange coupling between FM electrodes (Ji) .The foundational concept of this paper is based on the experimentally observed defects in the tunnel barrier of an MTJMSD Fig. . Data in20. It is critical to note that the density of defect scale with tunnel junction area. The same AlOx sample showed an increased number of defects after heating by the high-energy electron beam in TEM  between the top and bottom electrodes, is inevitable.To provide insights about the role of representative defects in MTJMSD here, we provide microscopy images collected on a system of cobalt  under ~ 2 nm AlOx insulator Fig. g. The deJi, we mainly studied MTJ with SiO2(substrate)/Ta(2 nm)/Co (5–7 nm)/NiFe(5 nm)/AlOx(~ 2 nm)/NiFe(10 nm) thin-film configuration. The shape of cylinders was defined photolithographically. We deposited all the metallic layers by a sputtering process using our optimum fabrication process reported elsewhere18.We performed Ferromagnetic Resonance (FMR) to investigate the effect of the defects in an MTJ before and after establishing molecular channels along the edges. For this study, we mainly utilized samples with several thousand cylindrical-shaped ~ 4 µm diameter MTJs. A large MTJ population produced strong FMR signals enabling unambiguous FMR mode detection from the as-received data. There are innumerable possibilities in MTJ types; however, to get the gist of defect induced 37. Coupled ferromagnets typically produced two modes: acoustic resonance mode (FMR mode with higher intensity) and optical resonance mode (FMR Resonance mode with lower intensity)37. MTJ with good quality tunnel barrier, presumably with structural properties similar to the micrograph . Defects producing net ferromagnetic coupling are not expected to show stable current suppression.We have provided several intriguing observations resulting from molecule-induced strong antiferromagnetic coupling elsewhere2 junction area and ~ 2 nm AlOx produce less than 0.5 µA current at 100 mV bias. An MTJ showing 10–100 times higher leakage current under identical conditions signifies the dominant presence of defects within ~ 2 nm tunnel barrier  and paramagnetic molecule-induced exchange coupling with two ferromagnetic electrodes Fig. This paper has included and discussed the transport study to highlight the impact of defect-induced coupling on MTJMSD  provide the opportunity to understand a vast range of MTJMSD that can be produced by combining various types of MTJ and paramagnetic molecules with uncertain amount and nature of defects. We conducted MCS to investigate the effect of defect-induced Ji, without delving into the nature of defects. We varied Ji for different cases of molecule-induced exchange coupling. The positive sign of Ji indicated ferromagnetic coupling, and the negative sign indicated antiferromagnetic type coupling.To understand the impact of defect-induced exchange coupling on MTJMSD, we performed Monte Carlo simulations (MCS). MCS studies investigating the effect of a wide range of Ji on the MTJMSD evolution process to equilibrium state; in this study, we maintained strong molecule-induced exchange coupling and kT = 0.1. To highlight the impact of Ji here, we mainly report MTJMSD, where molecule produced strong antiferromagnetic coupling. Molecule produced a net antiferromagnetic coupling when the sign of JmL and JmR was opposite, and magnitude was equal to 1. We recorded the magnetic moment of MTJMSD during the time span for which it evolved from random spin orientation to the equilibrium state , we investigated the 3D lattice model of MTJMSD . On the other hand, the difference between the color of molecules and the second ferromagnetic electrode is because the molecule made antiferromagnetic coupling with the second electrode (JmL = − 1).To further investigate the competing MSD Fig. . A 3D laule Fig. a. In thiule Fig. a. The clJi opposed the exchange coupling caused by molecules, the magnetic movement of the molecules was random . Hence, we conclude that even though molecules may be more ordered, they are still impacted by the defect-induced strong exchange coupling.When dom Fig. b. It is dom Fig. c, Y  to calculate the range of correlated interaction between the molecule and ferromagnetic state. The correlation factor was calculated by computing the dot product of the spin vector of each atom on an electrode with the average vector of the molecular analogs. The c factor was between − 1 to 1. The c = 1 represented the strong correlation and the case when molecules and ferromagnet are parallel. Whereas c = − 1 indicates that the molecules and ferromagnet are antiparallel.The impact of molecular spin states in different zones of the MTJMSD under the competing influence of molecular coupling and direct coupling is critical in defining the overall transport attributes. We calculated a correlation factor  complemented the impact of molecules induced strong antiferromagnetic coupling   appeared disjointed with the right electrode without the support of direct exchange coupling. The left electrode possessed regions corresponding to c in the 0.6 to − 1 range. Interestingly, when Ji was opposite to molecule-induced antiferromagnetic exchange coupling effect, i.e., JmL = − 1, and JmR = 1, direct coupling destroyed the molecule correlated states. Uncorrelated molecular and ferromagnetic electrodes are consistent with the 3D lattice images shown in Fig. For ero Fig. a. Howevedes Fig. b. In theion Fig. b. Howeveles Fig. b. For Jipin Fig. c. It is ate Fig. c. Howeve 1) Fig. d. Right  1) Fig. d. In ourJi), as discussed in Supplementary Fig. It is noteworthy that many possibilities may arise for a different order of magnitude of direct coupling and molecule-induced exchange coupling. However, due to limited space, a wide-ranging discussion is beyond the scope of this paper. However, we have investigated the change in energy of MTJMSD and interfaces to explore the nature of transitions  and direct coupling (Ji) to cover a wide range of possibilities. To understand the impact of the varying magnitude of Ji, we investigated the equilibrium energy of MTJMSD's Heisenberg model and interfaces as a function of the variation in molecular coupling strengths (JmL and JmR). The MTJMSD overall energy depends on the combined impact of molecule-induced exchange coupling and direct exchange coupling  to the opposite type. Typically, MTJMSD energy kept increasing as Ji approached near-zero magnitudes. In the absence of molecule-induced exchange coupling (for JmL = JmR = 0), MTJMSD's energy was symmetrical around Ji = 0. For stronger molecular coupling after JmL = JmR =  ~ 0.2Ji, as compared to ferromagnetic type Ji . Importantly, the energy reduction over JmL = JmR = 1, the amount of energy reduction necessary to reach the equilibrium state was ~ 5 units and ~ 40 units for Ji is opposite to molecular coupling nature, then MTJMSD energy will be higher as compared to the case when the nature of Ji and molecular coupling was the same, (ii) Ji also try to reduce the energy of the MTJMSD similar to a molecular coupling, (iii) as a key message we note that Ji ~ − 0.2 can influence ~ five times stronger ferromagnetic molecular exchange coupling effect.To demonstrate the direct exchange coupling Ui = 1) and antiferromagnetic (Ui = − 1) exchange coupling was determined. For the case of molecule-induced antiferromagnetic coupling (Ui = − 1), the interfacial energy kept increasing from − 1 to 0.2 and then decreased. A similar trend in the form of mirror image was observed for the molecule-induced ferromagnetic coupling  has to be around ~ 1/5th of molecular exchange coupling to overcome the molecular exchange coupling impact on MTJMSD. We also studied the effect of Ji on the molecule-ferromagnet interface for the case of molecule-induced antiferromagnetic coupling  on the impact of direct coupling and molecular coupling , the antiferromagnetic molecular coupling led to a low magnetization state around kT = 0.1. Increasing kT created thermal disturbance destroying molecular coupling impact and finally leading to a randomized state yielding nearly zero magnetic moments  for the ferromagnetic coupling regime  , MTJMSD's magnetic moment was generally low for all the cases of molecular coupling strength at low temperature   Fig. b. The MTnge Fig. b. Similaure Fig. c. The lo0.1 Fig. c and con.8) Fig. b. A simirgy Fig. b. Such r= 0 Fig. a and Ji − 1 Fig. c. We surJi = 0, the molecular magnetic moment was symmetrical around zero molecular coupling strength . Transport via defects expected to create hot spots for very high transport. Transport via defects also modifies the nature of defects36 and increases defect populations resulting in a failed MTJMSD. Future MTJMSD focusing on spin-transfer torque mechanism, where transport via MTJ is necessary, must ensure that coupling via defects is minimized to produce stable devices.The theoretical discussion in this paper is relevant to our experimental observations and will impact the future application of MTJMSD showing high TMR. Experimentally we observed that applied current change on a stable MTJMSD, where molecule induced strong exchange coupling, generally impacted the current state and promoted high current stateJi =  ~ 0.2 sufficient to disturb the five times stronger molecule induced strong antiferromagnetic coupling suggesting that the low magnitude of Ji can defeat the strong molecule coupling effect. This paper also indicates that an MTJMSD does not have to have the perfect insulator to produce molecular devices. MTJMSD with JiJi strength, molecular nanostructures can maintain an ordered state for low thermal energy. Future studies will focus on investigating the effect of Ji in the presence of other magnetic interactions and magnetic electrode properties. In future work, we also plan to focus on tunneling magnetoresistance (TMR) properties of MTJMSD based on our experimental and theoretical understanding of the role of defects.This paper focused on the molecule-induced exchange coupling in the presence of direct coupling via space between the two ferromagnetic electrodes. We reported experimental observations of stress-induced pinhole and hillocks type defects in the alumina (AlOx) tunnel barrier. Other tunnel barriers like MgO may have point defects. As a significant conclusion, every tunnel barrier for MTJMSD is expected to have some form of imperfection. We have demonstrated FMR as a tool to identify the presence of defects. Defects within tunnel barriers may lead to an inter-electrode exchange coupling competing with the molecular channels. Direct coupling between ferromagnetic electrodes either supported or canceled the impact of molecule-induced exchange coupling. We performed an MCS study to understand the impact of defect-induced exchange coupling on molecule-induced exchange coupling. The effect of defects was dependent on the thermal energy and molecular coupling. As thermal energy decreased, the impact of defects decreased, and molecules could still show the effect. Molecules in MTJMSD could still maintain correlated states at lower thermal energy. The mechanism behind the impact of direct exchange coupling was based on destroying the coherent molecular spin orientation in MTJMSD. We observed that Supplementary Information.


Saccharomyces cerevisiae and from shrimp shells, respectively. To evaluate the effect of each active ingredient on the nanofibers’ morphological features and bioactivity, nanofibers with both βG and CHI, only βG, only CHI and only copolymers, polyethylene oxide (PEO) and hydroxypropylmethylcellulose (HPMC) were fabricated. All four nanofiber formulations were also prepared with 1% CAM. The needle-free NanospiderTM technique allowed for the successful production of defect-free nanofibers containing all three active ingredients. The CAM-containing nanofibers had a burst CAM-release and a high absorption capacity. Nanofibers with all active ingredients  showed a concentration-dependent anti-inflammatory activity, while maintaining the antimicrobial activity of CAM. The promising anti-inflammatory properties, together with the high absorption capacity and antimicrobial effect, make these multifunctional nanofibers promising as dressings in local treatment of infected and exuding wounds, such as burn wounds.An active wound dressing should address the main goals in wound treatment, which are improved wound healing and reduced infection rates. We developed novel multifunctional nanofibrous wound dressings with three active ingredients: chloramphenicol (CAM), beta-glucan (βG) and chitosan (CHI), of which βG and CHI are active nanofiber-forming biopolymers isolated from the cell walls of  Infections are one of the main local causes of impaired wound healing, leading to increased morbidity . Wound iFor an optimal topical antibiotic therapy, an efficient delivery system with the embedded antimicrobial agent(s) is crucial . In thisTM technique with a stationary electrode wire used in this study. This technology offers several benefits (i) a continuous fiber production, (ii) it is faster than the traditional needle-electrospinning technique, and (iii) it might allow industrial-scale production of nanofibers [P. aeruginosa and E. coli [The most convenient way to produce nanofibers is by needle-electrospinning. This simple and flexible technique produces nanofibers from different polymers and offers several possibilities to tune the morphology and function of the spun nanofibers . Howevernofibers ,23,24,25nofibers  and the nofibers . Anothernofibers , which m E. coli . However E. coli . A commo E. coli .®). SBG® contains 2.5% βG (w/w) in water and forms a gel at room temperature [® is originally extracted from the cell walls of Saccharomyces cerevisiae, and is thereafter purified and hydrolysed by a patented method to obtain water-soluble βG with a mean MW of 7 × 105. SBG® has previously been proven beneficial as an active ingredient in the topical treatment of diabetic foot and leg ulcers [®-nanofibers successfully with the same technology as the one applied in this study [® but also with antimicrobial potential, making the nanofibrous wound dressing suitable for the treatment of infected wounds. The antimicrobial activity was targeted based on the known antibiotic CAM, as well as a second bioactive polymer, chitosan (CHI), exhibiting intrinsic antimicrobial activity. In this way, the SBG®-nanofibers, proven to have high wound healing potential in diabetic mice [S. aureus [Both natural and synthetic polymers can be used in electrospinning . Naturalperature ,32. The g ulcers . Grip etis study . We aimetic mice , in additic mice . CHI-hyd. aureus ,36. CHI . aureus , while B. aureus .Although both CHI and βG have been individually electrospun into nanofibers before, the combination of both polymers in one nanofibrous dressing is novel. To our knowledge, this is also the first study investigating the incorporation of CAM via electrospinning into nanofibers containing either CHI or βG. Furthermore, the number of studies published on needle-free electrospinning is still limited ,39. MoreTM. Toxic solvents are not only bad for the safety of the operator and the environment but might also affect the safety of the nanofibers negatively if solvent residues are not removed from the fibers [Since the spinnability of natural polymers is often poor, especially for CHI, co-polymers are often applied in electrospinning to improve the spinnability and mechanical properties of the fibers . We chose fibers .This study used a novel combination of active ingredients, combining CHI, known for its antimicrobial and wound healing properties, with the immunostimulating βG and the antibiotic CAM. Hereby, we aimed at developing a multifunctional nanofibrous wound dressing with a novel combination of features with known relevance for wound healing, exhibiting both antimicrobial and anti-inflammatory activity. To judge its appropriateness in wound healing, we determined features relevant to wound dressing, such as mechanical strength, swelling index and CAM release. Finally, we also tested cell toxicity and antimicrobial and anti-inflammatory activity in vitro.®; 2.5% (w/w) aqueous hydrogel) were a generous gift from Chitinor AS  and Biotec BetaGlucans AS , respectively. Hydroxypropylmethylcellulose (HPMC) (BenecelTM E4M HPMC) was obtained from Ashland . Polyethylene oxide (PEO), with an average molecular weight of 900,000 g/mol was manufactured by Dow Chemical Company . Albunorm®  was produced by Octapharma AG . Cell-counting kit-8, RPMI-1640 medium with L-glutamine and sodium bicarbonate, fetal bovine serum (FBS), penicillin-streptomycin, acetic acid (<99.9%), lipopolysaccharides (from Escherichia coli O55:B5), sulphanilamide, N-(1-Naphtyl)ethylenediamine dihydrochloride, ethanol (96% v/v), chloramphenicol (≥98%), sodium chloride, sodium phosphate dibasic dihydrate (Na2HPO4 · 2H2O) and potassium phosphate monobasic (KH2PO4) were purchased from Sigma-Aldrich . Ortho-phosphoric acid ≥85% was acquired from Kebo Lab Ab . Dulbecco’s Modified Eagle Medium (DMEM) High Glucose was purchased from Biowest . Chloramphenicol Antimicrobial Susceptibility discs  were acquired from Thermo Fisher Scientific Inc. . Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and murine macrophages (RAW 264.7 cells) were obtained from ATCC . Human keratinocytes (HaCaT cells) were acquired from AddexBio . The substrate used for electrospinning (polypropylene blue spunbond) was delivered by Elmarco .Chitosan (CHI) from shrimp  and soluble beta-1,3/1,6-glucan (βG) (SBGw/w). All polymer solutions were prepared with the same solvents, with a final concentration of 60% (w/w) ethanol, 3% (w/w) acetic acid and 34.5% (w/w) water. The polymer composition, as well as the CAM-content of the different polymer solutions, is presented as % dry content in ®, water and acetic acid and heated on a magnetic stirrer overnight at 50 °C. Since PEO is soluble in ethanol when heated [w/w) PEO was first dispersed in ethanol, and further dissolved under constant stirring at 70°C for at least 80 min. The 5% PEO solution and the βG and/or CHI-containing solution were added to an HPMC-dispersion in ethanol. The amount of HPMC and ethanol was adjusted to achieve a final ethanol concentration of 60% (w/w) and the PEO content wanted for the specific formulation . Surface tension was determined applying the Du Noüy ring method with a Pt-Ir-ring K 610  using a K6 force tensiometer .Conductivity of the polymer solutions was measured using a Sension® Rotavisc hi-vi II Complete , using the spindle DIN-SP-6 with the sample holder DIN-C-2 . The measurement time was one minute with a rotational speed of 20 rpm. The viscosity was recorded as a function of the shear stress over the shear rate.The viscosity of the solutions was measured applying the IKAThe pH was examined using a Fisherbrand™ accumet™ AP115 Portable pH Meter Kit . All measurements were performed in triplicates.TM NS Lab machine , equipped with a stationary spinning electrode wire with an applicable spinning voltage of 80 kV DC current with a unidirectional substrate unwind/rewind. The substrate speed was 2 mm/min and the wire-to-collector distance was kept at the maximum distance of 24 cm. A one-sided carriage (40 mL), filled with the polymer solution, coated the electrospinning wire at its maximum speed of 300 mm/s. All solutions were spun for 80 min in total. The carriage was refilled with polymer solutions every 20 min. The relative humidity was in general targeted to be between 24 and 29%, but one out of three replicates was spun at a higher humidity of 39 and 40% for two formulations (βG-CAM-nf and CHI-CAM-nf), respectively. The temperature was kept at 24 ± 2 °C. For analysis, the nanofibrous mat was always removed from the substrate.The polymers solutions were electrospun using the Elmarco NanospiderMorphology of the nanofibers was examined by Field Emission Scanning Electron Microscopy (FE-SEM) using the Zeiss Sigma FE-SEM microscope . The specimens were dried in a desiccator overnight prior to measurement. A double-sided carbon tape was applied to mount the specimens on the stubs before coating with gold/palladium using a Polacron SC7640 high-resolution sputter coater . All samples were taken from the middle of the nanofibrous mat. Fiber diameter was analysed using ImageJ . Three pTensile properties of the nanofibers were investigated applying the Texture Analyzer TA.XT plus . The method was based on the ASTM-Standard D882-18 . The thi3, 0.30 g/L KCl, 0.37 g/L CaCl2·2H2O and 165 mL/L Albunorm®. This composition was slightly modified from a previously described simulated wound fluid, as we used human serum albumin (Albunorm®) instead of bovine albumin [I, A). The experiment was repeated three times per formulation and the swelling index was calculated using the following Equation (1):I: Initial weight specimenWA: Weight of specimen after swellingWSwelling index of the nanofibers was tested using simulated wound fluid containing 5.84 g/L NaCl, 3.60 g/L NaHCO albumin . Fiber s2. A cellophane membrane  was used to separate the acceptor and donor compartment as previously described [2PO4, 2.98 g/L Na2HPO4·2H2O, 8 g/L NaCl) that was kept at a constant temperature of 32 °C. Round fiber samples with a diameter of 9 mm were cut, weighed  at λ = 278 nm.Drug release from nanofibers containing CAM  was investigated using jacketed Franz diffusion cells  with a 5 mL acceptor chamber and a donor diffusion area of 0.64 cmescribed . The acc weighed  and placv/v) FBS and penicillin-streptomycin. Cells were seeded on 96 well plates  and incubated for 24 h at 37 °C in 5% CO2. Nanofibers were dissolved in 0.1% acetic acid (w/w) in Milli-Q-water at a concentration of 10 mg/mL and diluted with medium for further sample preparation. The cells were treated with 10 µL medium (control) or 10 µL nanofiber solutions (samples), corresponding to final fiber concentrations of 125, 250 and 1000 µg/mL. After further incubation for 24 h, CCK-8 reagent (10 µL) was added to each well and the plates were thereafter incubated for 4 h. Absorbance was measured at λ = 450 nm with a reference at λ = 650 nm using a Spark® multimode microplate reader . Each sample-concentration was tested in triplicate. The relative cell viability was calculated using the following Equation (2):S: Absorption of the samplesAC: Absorption of the controlAThe in vitro cytotoxicity of the nanofibers was tested in both human keratinocytes (HaCaT cells) and murine macrophages (RAW 264.7) using the Cell Counting Kit-8 . HaCaT cells and RAW 264.7 cells were cultured in DMEM and RPMI-medium, respectively. Both media were supplemented with 10% (Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), representing Gram-negative and Gram-positive bacteria, respectively, was determined using a modified disc diffusion assay, as described previously [Antibacterial activity of nanofibers against eviously . Bacteri5 cells/mL) were seeded on 24-well plates (1 mL/well) and incubated at 37 °C in 5% CO2. After 24 h incubation, RPMI medium was replaced with 990 µL LPS-containing medium (1 µg/mL) and 10 µL of the dissolved formulations, corresponding to three different fiber concentrations . Untreated cells were used as control. The cells were further incubated for 24 h. The NO production was measured by addition of Griess reagent  in a ratio of 1:2 to samples from the cell supernatant. The absorption was measured on a Spark® multimode microplate reader  at λ = 540 nm. The reduction in NO production was assumed to reflect the anti-inflammatory activity of the formulations on the cells. The anti-inflammatory activity was thus calculated as the NO production in cells treated with the formulations compared to untreated cells, using the following Equation (3):S: Absorption of the samplesAC: Absorption of the controlAThe anti-inflammatory activity of the nanofiber formulations was evaluated by measuring the NO production in LPS-stimulated murine macrophages (RAW 264.7) . Samplesp-value < 0.05 was considered statistically significant.One-way ANOVA followed by a Tukey test using GraphPad Prism  was used to test for statistical significance. A TM technology. Several variables, among them the solution composition and properties, ambient conditions and spinning settings, are known to influence the electrospinning process and nanofiber morphology [w/w). The solvent system, regarded as suitable for solubilising all components, as well as being eco-friendly, consisted of ethanol, acetic acid and water. The standardisation of the experimental setup was performed in preliminary experiments (results not shown).The first part of this study focused on the preparation of suitable polymer solutions for electrospinning using the needle-free electrospinning Nanospiderrphology . We therTM, since a high viscosity might limit the gravidity-dependant feeding rate of the electrospinning wire [TM system might be different. Thus, we focused on their effect on the morphology of the obtained nanofibers.Important features of polymer solutions used in electrospinning are the surface tension, conductivity and viscosity, as these characteristics are known to influence both the spinning process and quality of the formed fibers . The poling wire . In addiing wire . The infing wire ,51,52,53w/w)). The addition of CAM did not affect the solution properties (The characteristics of the polymer solutions are summarised in operties .All polymer solutions were spun applying the same settings (described in w/w) dry material. Although it has been reported previously that a surface tension above 42 mN/m was needed for the formation of beadless PEO/Curdlan fibers by needle-electrospinning [TM. The lower surface tension required in needle-free electrospinning is in accordance with previously published data: Ramakrishnan et al. examined the effect of solution parameters in needle-free electrospinning and found that a low surface tension (32 mN/m) improved the quality of PEO-nanofibers [The morphology and diameter distribution of CAM-free and CAM-containing nanofibers obtained from the different polymer solutions is shown in spinning , we expenofibers .w/w) CAM. This was expected since polymer solutions with and without CAM showed similar properties  and 21.8 MPa (βG-nf), no significant difference in tensile strength could be seen between the different formulations due to the high standard deviations . The addIt is critical to maintain a moisture balance in the wound, both for an optimal wound healing, and to limit the scar formation ,59. PolyIn contrast, nanofibers containing CHI  showed a higher stability upon contact to fluid. These nanofibers could therefore be withdrawn from the media after 5 min. These fibers were found to have a high swelling index between 779 and 1055% . As swelA high swelling index is linked to several beneficial features of a wound dressing, as a wound dressing should instantly attach to the wound and adapt to its shape to assure a tight barrier, protecting against bacterial invasion and allowing the best possible tissue contact . In addiAll nanofibers in this study swelled to a high degree and formed a transparent gel, suggesting that they will immediately attach to a wound, adjust to its shape and allow the examination of the wound without the need to remove the dressing due to its transparency.The release kinetics of the antibiotic ingredient, in this case CAM, will affect the antimicrobial effectivity of the dressing. For successful treatment of infections, a therapeutically active concentration of the antibiotic agent(s) needs to be present at the wound site. The in vitro release of CAM from nanofibers was tested for 6 h in a Franz diffusion setup. The CAM-release is shown in The burst drug release from nanofibers was expected, taking into account (i) the thin fiber diameter of between 99 to 150 nm , (ii) thAn ideal dressing should, amongst other features, be biodegradable and biocompatible . The cytE. coli and S. aureus as representative gram-negative and gram-positive bacteria, respectively. Both these pathogens are commonly found in infected burn wounds [Infections are one of the leading causes for death after burn injuries; therefore, the use of a dressing that has antimicrobial activity is favourable . We fabrn wounds .E. coli nor for S. aureus ) and most of the acetic acid evaporated during the electrospinning process, resulting in fibers with a neutral pH when applied on the agar plate (pH measurements not shown). This implies that CHI is not protonated, and thus does not have the aforementioned antimicrobial effect. Similar observations have been described previously, for CHI films containing 2% (w/v) CHI, prepared in 2% (v/v) acetic acid solution [E. coli, S. aureus and S. epidermidis. On the contrary, Abid et al. showed an antimicrobial effect from fibers spun with a solvent containing 50% acetic acid [CHI is known to have antimicrobial properties and Abid et al. reported that CHI-PEO fibers showed zones of inhibition of 22.3 ± 3.0 nm and 14.7 ± 1.36 nm (over 24 h) for ectively . Howeverectively . Our nansolution . Here, ntic acid . Thus, aThe occurrence of severe burns in combination with the following surgical actions can lead to an uncontrolled inflammatory reaction . AlthougAnti-inflammatory activity of both βG and CHI in macrophages has been reported previously ,76. The We found a significant concentration-dependent inhibition of NO production for nanofibers containing both βG and CHI (βG-CHI-nf and βG-CHI-CAM-nf). At the same time, nanofibers formed from only the co-polymers did not influence the NO production and showed no anti-inflammatory activity. Nanofibers containing only one of the active polymers (βG or CHI) had just a slight and not significant reduction in NO production with increasing concentration . The lowSince the anti-inflammatory effect of nanofibers containing both active polymers (βG-CHI-nf and βG-CHI-CAM-nf) was higher than the combined decrease in NO production from the nanofibers containing only βG (βG-nf and βG-CAM-nf) or CHI (CHI-nf and CHI-CAM-nf), a synergistic effect is suggested when combining these two active biopolymers in the same dressing. It seems like the low concentrations of βG and CHI tested separately did not affect the macrophages while the combination of the two ingredients led to a concentration that was high enough to modulate the anti-inflammatory response of macrophages, thus leading to a reduction in NO production. The production of NO is an indication of inflammation in macrophages . NO has In this study, we fabricated nanofibrous wound dressings with three different active ingredients, βG, CHI and CAM. The formulation combining all three active ingredients (βG-CHI-CAM-nf) showed anti-inflammatory properties  as well ®) and/or chitosan, and with or without 1% (w/w) chloramphenicol, were successfully prepared by needle-free electrospinning, and their morphology examined by FE-SEM. All formulations formed uniform nanofibers with a diameter of approximately 100 nm. Among the tested formulations, was the formulation containing all three active ingredients; beta-glucan, chitosan and chloramphenicol, judged the most promising, supported by the in vitro anti-inflammatory activity observed in macrophages, which was higher for this formulation compared to the other formulations. Furthermore, the antimicrobial activity of chloramphenicol was well-maintained in all nanofibers, demonstrated by the disc diffusion assay with two bacterial strains: E. coli and S. aureus. These characteristics, as well as the in vitro biocompatibility of the formulation in HaCaT and RAW 264.7 cell lines, make these dressings promising for infected wounds and wounds with a high inflammatory reaction.Nanofibrous wound dressings comprising the bioactive polymers; soluble beta-glucan (SBG


The use of devulcanized tire powder as an effective reinforcement in self-healing styrene-butadiene rubber (SBR) compounds has been investigated for the first time in this work. For this purpose, the evolution of the microstructure of the rubber from end-of-life tires (ELTs) was studied during granulation, grinding and devulcanization through an exhaustive characterization work in order to relate the final microstructure with the mechanical response of the repaired systems. Different morphologies (particle size distribution and specific surface area) obtained by cryogenic and water jet grinding processes, as well as different devulcanization techniques , were analyzed. The results demonstrated the key influence of the morphology of the ground tire rubber (GTR) on the obtained devulcanized products (dGTR). The predictions of the Horikx curves regarding the selectivity of the applied devulcanization processes were validated, thereby; a model of the microstructure of these materials was defined. This model made it possible to relate the morphology of GTR and dGTR with their activity as reinforcement in self-healing formulations. In this sense, higher specific surface area and percentage of free surface polymeric chains resulted in better mechanical performance and more effective healing. Such a strategy enabled an overall healing efficiency of more than 80% in terms of a real mechanical recovery (tensile strength and elongation at break), when adding 30 phr of dGTR. These results open a great opportunity to find the desired balance between the mechanical properties before and after self-repair, thus providing a high technological valorization to waste tires. Tires are mandatory products for the mobility of people and goods. However, these vital elements are not capable of being recycled due to their complex structures. Nowadays, the amount of waste tires discarded worldwide is approximately 800 million units, 10 million tons per year. If one considers that the amount of natural and synthetic rubber in tires is about 60%, 6 million tons of rubber from tires are disposed each year . In thisSome of the previously considered solutions for the disposal of ELTs  have become less and less viable because of the associated environmental problems and high recovery costs. Instead, the reuse and valorization of the constituent materials have become the preferential solution. Methods and technologies that transform ELTs into raw materials primarily depend on the use of the tire  since each application involves different rubber compounds. These technologies are grouped into three levels ,4,5, folThe main current challenge is related to improving the efficiency of the abovementioned technologies and to transforming ELTs into new raw materials and products with high added value. In line with this objective, research in which, for example, recycled fibers from car tire waste were used to successfully develop a rubber aerogel, has been carried out. Aerogels are ideal for many high-value applications such as drug-delivery pharmaceuticals, filters for pollutants, and building insulation materials ,24,25. IThe work presented here is part of an ongoing research line that aims to develop elastomeric systems that can combine self-healing properties with the use of ELT rubber by acting on the tire rubber compound and using devulcanized rubber as reinforcement in self-healing matrices. We report the systematic and comparative microstructural analyses of diverse ground tire rubber (GTR) and devulcanized ground tire rubber (dGTR) originating from end-of-life truck tires ) and obtained by different methods and technologies, thus enabling the definition of a structural model related to the selectivity of the applied devulcanization process. This is intended to demonstrates the possibilities of both the secondary raw material and the available recycling levels  used to valorize rubber from ELTs. Accordingly, self-healing SBR compounds filled with GTR and dGTR were prepared and characterized. These results were used to establish a relationship between the microstructures of GTR and dGTR and the self-healing efficiency and mechanical performance of SBR compounds. Finally, it is expected that this work will contribute to the perception of the recycling of ELTs as not only a benefit for the environment and the economy but also an opportunity for scientific-technological progress.Rubber granules from end-of-life truck tires were used. Two grinding technologies were further employed: cryogenic grinding and water jetting. Lehigh Technologies supplied cryogenic GTR (GTR-Cryo) and Rubber Jet supplied the GTR obtained with water jet technology (GTR-WJ), both within the same particle size range. Details on the particle size and composition of the as-received departure material (granules) and the resulting ground material (powder) are reported in Three robust devulcanization techniques, based on different principles, were chosen to devulcanize the GTR powder samples. The procedures are described below.TM devulcanization was carried out in an internal mixer  with a filling volume of 70%, using Banbury-type rotors at room temperature and a rotor speed of 30 rpm for 10 min. Although the equipment was initially at room temperature, friction heated the material. As a result, the average process temperature was 70 °C.Microwave devulcanization was carried out in a microwave prototype ,33. SampBis(3-triethoxysilyl propyl) tetrasulfide (TESPT) was used as devulcanizing agent following previously reported procedures ,34,35. GStyrene-butadiene rubber (E-SBR Europrene 1502) and commercial-grade vulcanizing additives supplied by Sigma-Aldrich  were used as-received. Mixing was performed in an open two-roll mill  at room temperature using a rotor speed ratio of 1:1.5. First, rubber was passed through the rolls until a band was formed. The activating complex (zinc oxide (ZnO) and stearic acid (SA)) and filler (GTR or dGTR powder) were then progressively added to the rubber; finally, the curatives (N-cyclohexylbenzothiazole-2-sulphenamide (CBS) and sulfur (S)) were added.c = 160 °C, frequency of 0.833 Hz, and 2.79% strain for 60 min. The composites were then vulcanized in an electrically heated hydraulic press  at 160 °C and 200 MPa according to their t90, as derived from the corresponding curing curves  at curing temperature Trves see . SamplesAbout 5 g of GTR or dGTR were extracted (Soxhlet extraction) in acetone for 24 h and subsequently extracted in toluene for 72 h. After extraction, each sample was dried at 45 °C until a constant weight was reached. The sol fraction for each of the extractants was calculated with Equation (1).The sol fraction was defined as the sum of the soluble fractions in acetone and toluene.ν) in the mass-basis form, which is the number of moles of sulfur crosslinks per unit mass of rubber, was determined through equilibrium swelling experiments. About 0.2 g of the acetone-extracted powder sample were placed in toluene at room temperature and in darkness—to avoid molecular changes—for 72 h. Analyzing samples in powder form without any additional processing avoids possible alterations of the crosslink density . M. M34]. MQuantitatively, we determined the healing efficiency based on two parameters, the recovery of the elongation at break and of the tensile strength, as illustrated in The second aspect worth analyzing is the effect of the devulcanization on the healing capability of SBR compounds. From This study explored the effect of free polymeric chains on the surface of partially devulcanized recycled tire powder (from end-of-life truck tires) in self-healing SBR compounds. The importance of understanding the evolution of the microstructure during the different levels of the recycling of ELTs  and devulcanization (dGTR)) has also been demonstrated via the establishment of relationships between particle shape, composition, and specific surface area with the efficiency of various devulcanization processes. First, we found that the microstructure of the departing GTR influenced the obtainment of devulcanized products (dGTR), affecting the decrease in the network density and selectivity. Secondly, the thermo-mechanical devulcanization (TM) provided the highest amount of free long chains on the surface of the ground powder, regardless of the type of departing grinding method (cryogenic or WJ). This devulcanization technique also proved to be the most selective crosslink scission method when dealing with end-of-life truck tires.A selected content (30 phr) of GTR and TM-dGTR particles was further added to a self-healing SBR compound. The morphological and dynamic features of the grains suggested that the key factors leading to the recovery of the mechanical properties are the high percentage of free surface polymeric chains and high devulcanization selectivity, achieving a healing efficiency of more than 80% based on the recovery of both stress and strain. In conclusion, the research discussed here gathered information at different levels (molecular and micro/macro), and it presents a good phenomenological approach for a better understanding of the underlying healing mechanism taking place in elastomeric materials. Future work will deal with the optimization of devulcanized particle content, as well as the incorporation of GTR/dGTR in filled SBR compounds, in the search of better mechanical properties to be scaled up to real-life applications.


We report five cases of sudden intrauterine death due to premature closure of the ductus arteriosus. In four cases, this was caused by dissecting the hematoma of the ductus arteriosus with intimal flap and obliteration of the lumen. In one case, the ductus arteriosus was aneurysmatic, with lumen occlusion caused by thrombus stratification. No drug therapy or free medication consumption were reported during pregnancy. The time of stillbirth ranged between 26 and 33 gestational weeks. We performed TUNEL analysis for apoptosis quantification. The dissecting features were intimal tears with flap formation in four of the cases, just above the origin of the ductus arteriosus from the pulmonary artery. The dissecting hematoma of the ductus arteriosus extended downward to the descending aorta and backward to the aortic arch with involvement of the left carotid and left subclavian arteries. TUNEL analysis showed a high number of apoptotic smooth muscle cells in the media in two cases. Abnormal ductal remodeling with absence of subintimal cushions, lacunar spaces rich in glycosaminoglycans , and smooth muscle cell apoptosis were the pathological substrates accounting for failure of remodeling process and dissection. Sudden intrauterine death is a socio-epidemiological problem. It accounts for 4–5/1000 births. The causes of unexpected death are mostly related to placenta injury in terms of placenta insufficiency, maternal malperfusion vascularization, inadequate fetal vascularization, or umbilical cord causes, such as nodal cord, rupture, marginal insertion, etc. . Most ofPremature closure of the ductus arteriosus is a rare condition, which is difficult to diagnose. The incidence is not well defined ,5,6,7,8.During fetal life, the ductus arteriosus, with its patency, guarantees blood flow from the right ventricle into the descending aorta bypassing the lungs since the role of blood oxygenation is played by the placenta.The fetal ductus arteriosus is a large muscular artery originating from the 6th aortic arch ,10,11, cTwo different conditions can affect the ductus arteriosus before birth: excessive patency with aneurysm formation and premature closure with lumen obstruction ,17.Aneurysm of the ductus arteriosus, a relatively rare lesion detected mostly in children and adults, can be incidental and asymptomatic but can evolve to spontaneous dissection, rupture, thrombosis, and embolism of the aorta and pulmonary arteries, leading to death ,23,24,25Aneurysms of the ductus arteriosus can be congenital (present at birth) or acquired. Congenital ductus arteriosus aneurysms are reported to be more common than originally thought postnatal and can remain asymptomatic at birth ,27,28.Several theories have been postulated to explain the mechanism of congenital aneurysm formation of the ductus arteriosus ,29,30. TThe persistent ductus arteriosus has been associated with chromosomal abnormalities in 10% of the cases and TGFBeta signaling abnormalities, as in Marfan or Loeys Dietz, to TFAP2B in Char syndrome and to TFAP2B, TNF-RAF1, prostacyclin synthase in premature and term infants ,34.Few case reports present dissecting aneurysms of the ductus arteriosus as a cause of sudden unexpected intrauterine death.The purpose of our study was to report the clinical and pathological characteristics of five cases of unexpected and sudden intrauterine death due to dissection of the ductus arteriosus and to elucidate the possible mechanism leading to this severe complication and ultimately to death.Since 2013, our center at Padua University has acted as a tertiary referral center in the Veneto region for sudden intrauterine death. All the unexpected and sudden intrauterine deaths of the Veneto region are referred to Padua for autopsy, which are performed by two expert cardiovascular pathologists on rotation . The autopsy was a complete autopsy performed according to a standard adopted protocol for fetal autopsy inclusive of radiographic and photographic documentation. Clinical data from the mother and the pregnancy are reported in the clinical information protocol attached to the autopsy request. This protocol includes the metabolic and infective profile of the mother together with the family history and the documentation of the pregnancy. Routinely, the standard protocol for autopsy foresees histology of all the organs and if the maceration is not too advanced, we store fresh tissues for genetic studies when requested.Among 375 cases collected since then, from 25 weeks of gestation to term, we have identified at autopsy, 5 cases (1.3%) of abrupt ductus arteriosus closure caused by dissection of the ductus with hematoma of the parietal wall and ab extrinseco compression of the lumen. We removed the thoracic block including the heart and the lungs and further evaluated them under a stereomicroscope. We used the sequential segmental approach for categorization. We opened the heart according to the blood flow. For histology, we sampled all the thoracic and abdominal organs. For the heart, we made a transverse cut to include the right ventricle, septum, and the left ventricle.The ductus was sectioned with a serial transverse cut every 3 mm from the pulmonary junction to the aortic end, with a mean of three paraffin blocks per case. For each macroscopic block, we performed hematoxylin–eosin, elastic fiber–van Gieson, and Alcian–PAS staining. For apoptosis assessment, we performed TUNEL staining.The protocol for sudden intrauterine death includes also the evaluation of the placenta according to the international guidelines “Sampling and definitions of placental lesions: Amsterdam placental workshop group consensus statement” .We present five cases of ductus arteriosus dissection, all male, as a cause of death (1.3%) from our registry of sudden intrauterine death since 2013. We used ReCoDe classification for causes of death  and accoPostmortem examination of the five cases.At autopsy, the external examination showed a normally developed fetus for the gestational week see . All theExamination of the thorax and abdomen also showed normally related organs with a preserved normal laterality and no associated pathologies. No congenital defects were observed anywhere. In the thorax, after opening the pericardium, in four cases, adventitial hemorrhagic imbibition/hematoma at the level of the ductus arteriosus, giving an appearance of dilatation exceeding the diameter of the aorta and the pulmonary trunk, suggestive of dissection/hematoma was identified. Such hematoma started at the pulmonary end of the ductus arteriosus, partially involving the pulmonary trunk, the entire length of the ductus arteriosus, and extending into the descending aorta but stopped immediately after the junction with the ductus arteriosus. There was also the involvement of the origin of the left subclavian and left common carotid arteries . The heaThe lungs were normally located, the right trilobed, supplied by short, epi-arterial bronchus, and the left bilobed, supplied by a long, hypoarterial bronchus. The sectioning of the lung revealed, as expected, a compacted parenchyma, which, at histology, showed a saccular pattern in cases 1, 2, 3, and 5 and a canalicular pattern in case 4, in keeping with the gestational age. No vascular intraparenchymal abnormalities were present.No emboli or infarcts were evident in the downstream circulation.The placenta was available in four of the five cases. We could not detect any sign of infections; in two cases (case 4 and 5), we identified multifocal small infarcts. In another case (case 2), we recognized maternal vascular malperfusion. The last placenta (case 1) was unremarkable.The microscopic examination of the ductus arteriosus showed absence of subendothelial intimal cushions with intact internal elastic lamina in three cases. In three cases, the presence of cystic medial necrosis (lacunar spaces in the media) were the prominent abnormalities . ApoptosThe results of our morphological study on five cases of stillbirth babies due to dissecting aneurysm of the ductus arteriosus showed that the dissection is similar to that affecting the aorta and the great vessel and other muscular arteries such as the coronary and cerebral arteries. We were able to identify an intima tear at the junction between the pulmonary trunk and the ductus with an intima-media flap and hematoma dissection through the media and the adventitia without any re-entry mechanisms in four of the five cases. This led to occlusion of the lumen by the flap segment and circumferential hematoma. In all the four cases, the hematoma dissection involved the descending aorta and the retrograde aortic arch. In only one case, we were unable to identify an intimal tear or parietal dissection, but the lesion was ascribed to ductal aneurysm and occlusive thrombus stratification.There are no differences in terms of morphological substrates if we compare the dissection of the aorta or the coronary arteries with the dissection of the ductus arteriosus ,38. In bDuring pregnancy, the ductus arteriosus remains open, and during the second trimester, the proliferation of intimal cushions with lumen reduction will prepare for ductus arteriosus closure. The lumen is stellate in the closing duct and is reduced in size, mainly in the pulmonary extremity ,15,24,25Remodeling for the preparation of ductus arteriosus closure at birth with immediate contraction of the parietal wall implies differentiation of vascular SMCs and endothelial cells, production of the extracellular matrix, vascular SMCs proliferation and migration, and finally, a decrease in elastic fibers and an increase in fibrous connective tissue ,25. The In the congenital aortic arch aneurysm, the ductus arteriosus is usually tortuous, saccular, and dilated and elongated, and characterized microscopically by a lack of intimal cushion formation and defective elastic lamellae formation, enhancement of necrosis, and mucoid degeneration of the inner part of the media ,15. ThisIn all the other 370 cases of our registry, we did not identify the presence of aneurysms of the ductus arteriosus. Dissecting aneurysm of ductus arteriosus can be regarded as the end of the spectrum of the patent ductus arteriosus (PDA), a congenital cardiac abnormality that can be a single cardiac abnormality or can be part of other associated congenital heart diseases. PDA is characterized by the lack of closure at the time of birth with the persistence of patency, allowing shunting of blood between the systemic and the pulmonary circulations .At a histomorphological analysis, the patent ductus arteriosus is characterized by the absence of the features at the base of remodeling for closure after birth, which are the intimal cushions, pads of proliferating SMCs migrating from the media into the intima, and thickening of the medial wall as a consequence of longitudinal SMCs fascicles arrangement in the subintimal space ,30,31. TDissecting aneurysm is an acute event that has led to fetal death. Since this was an unexpected and sudden event, we do not know the exact time frame for the dissection of the parietal wall, occlusion of the ductus arteriosus, and death. The barrage at the ductus arteriosus would have caused a redirection of flow toward the pulmonary arteries and an increase in blood shunt from the right atrium to the left atrium. The dimension of the foramen ovale would play a crucial role in the hemodynamics of these fetuses. Very few papers have reported dissection of the ductus arteriosus as the cause of sudden unexpected deaths in single-center experiences or as anecdotal cases without an accurate and detailed description of the parietal dissected wall ,41,42.One point should be discussed as well in relation to the terminology adopted in the literature. As in adults with aortic dissection, in which we are not utilizing anymore the term dissecting aneurysm, since the dissection has been recognized to occur in the absence of ectasia or aneurysm formation, it would be more appropriate to adopt the term dissection of ductus arteriosus rather than dissecting aneurysm. The dilatation is not a prerequisite for dissection, but dissection can manifest without an increase in the diameter of the vessel.On the other hand, an aneurysm of the ductus arteriosus can be present and be detected accidentally during the fetal ultrasound and resolved spontaneously after birth with closure without complications .Several theories have been proposed for the development of an aneurysm of the ductus arteriosus. At the time of birth, delayed closure of the aortic end of the ductus would expose the ductal wall to high pressure with related dilatation. During the third trimester, as detected by ultrasound, dilatation and aneurysm development has been ascribed to the weakening of the parietal wall with a reduction of intimal cushion formation or deposition of elastin. Aneurysm development in some reported cases was associated with defective fibrillin or collage ,41 or vaTo the best of our knowledge, a ductal aneurysm has never been described in the stillborn fetus.Spontaneous premature closure of the fetal ductus can be a cause of fetal demise. However, in the cases reported in the literature, no morphological description of the mechanism of closure has been described ,16,45,46Some other aspects are puzzling in our five cases. All of them were males. However, we do not know if this is by chance or it implies any male gender propensity to dissection. All of them except one occurred at around 32 gestational weeks, which is the time of the remodeling process of the arterial duct, with intimal cushion formation in preparation for physiological closure occurring at birth. This could imply failure of the remodeling process.The main limitation is represented by the poor quality of the autoptic material on which we performed the histological and genetic assessments, in part due to the severe maceration of four of the five cases. As a consequence of the high level of degradation of DNA, it was not possible to detect possible genetic collagenopathies. Indeed, these genes are characterized by long nucleotide sequences.In case of sudden unexpected intrauterine death, a thorough autopsy should be performed to identify rare conditions such as dissecting ductus arteriosus as the cause of death. Histological examination should be performed for the detection of pathological substrates accounting for intimal tears, dissection, hematoma, apoptosis, and absence of inflammatory infiltrate. Further genetic studies should be carried out to confirm or exclude the genetic background.


Plasma levels of IL-5, IL-6, and IP-10 are associated with recurrence of HIV-associated KS, while persistently detectable HIV viral loads increase the risk of KS recurrence.Kaposi sarcoma (KS) is an AIDS-defining angio-proliferative malignancy highly prevalent in Sub-Saharan Africa. The main objective of this study was to determine the factors associated with recurrence of HIV-associated KS. We recruited a cohort of individuals on antiretroviral therapy who were in remission for HIV-associated KS after undergoing cytotoxic cancer chemotherapy. Collected variables included sociodemographic and clinical parameters, cytokines and chemokines, HIV viral loads, and CD4 counts. Compared to individuals who had KS recurrence, IL-5 was significantly higher at time of follow-up in individuals who had sustained remission (22.7pg/ml  The vascular malignancy Kaposi sarcoma (KS) is highly prevalent in sub-Saharan Africa (SSA) . It is cThe two prevalent forms of KS in SSA include endemic KS which predominantly affects HIV-uninfected males of SSA, and epidemic or HIV/AIDS-associated KS which is a result of HIV-induced immunosuppression . HIV-assClinical outcomes to treatment of HIV-associated KS are variable, with poor treatment response and high disease recurrence rates observed previously. In a previous study, we found that only about 47 percent of HIV-associated KS patients undergoing first-line chemotherapy (ABV) had a complete response to treatment, while 53 percent had a partial response [Factors associated with KS disease re-development after initial successful treatment are poorly understood. However, several clinical, immunological, and virological factors have been associated with KS development and treatment outcomes. Presence of nodular lesions as opposed to patches and plaques have been observed to be associated with advanced disease and poor treatment response in KS patients . High plThe aim of this study was to determine factors that are associated with re-development of HIV-associated KS in individuals who were on ART and in remission for KS after previous successful treatment with chemotherapy. The factors that were investigated included clinical, sociodemographic, immunological (cytokines and chemokines), and HIV-related.This was a prospective study that recruited a cohort of individuals who had been previously successfully treated and were in remission for HIV-associated KS. These individuals were all HIV positive and on ART for at least 6 months at baseline. All these individuals were treated in an outpatient setting and had no history of visceral involvement. They had all been previously treated with the commonly available first-line chemotherapy regimen . At the time of recruitment, these individuals had completed at least 6 cycles of chemotherapy (given 3 weeks apart) and were in remission for KS, with no evidence of disease. They were followed up until they experienced KS recurrence, or for a maximum period of 12 months. The main objective of the study was to determine if and how cytokines/chemokines and/or HIV-related parameters are associated with recurrence of chemotherapy-treated HIV-associated KS.At baseline, sociodemographic and clinical information were collected. The patients were then followed up monthly in the first 4 months then at 6 months, then 9 months and then at 12 months to observe for any signs of disease recurrence. Patients also came in at unscheduled follow-up time points. The study follow-up was ended and second-line chemotherapy initiated for individuals that experienced KS recurrence during the follow-up period. Venous whole blood was collected in EDTA vacutainer bottles at baseline and at follow-up visits. Plasma was separated from the whole blood and stored at -80°C until analysis. For this study, analyzed samples included those collected at baseline and at time of KS recurrence for individuals who experienced KS re-development, and at 3 months of follow-up (post-remission) or the latest follow-up (up to 12 months) for those who had sustained remission. This study was approved by the University of Zambia Biomedical Research Ethics Committee (Ref. No. 019-07-18) and the Zambian National Health Research Authority. Written informed consent was obtained from each study participant before enrollment into the study.We performed HIV viral load at baseline and at the follow-up time point. The HIV-1 plasma viral load was measured using Aptima HIV-1 Quant Dx Assay kit on the Hologic Panther (Hologic) according to the manufacturer’s protocol.CD4 counts were performed at baseline and at the follow-up time point. The BD TriTest kit (BD biosciences) was used according to the manufacturer’s protocol, and counts were determined on a BD FACSCalibur (BD Biosciences).Plasma cytokines and chemokines were quantified using the MILLIPLEX MAP human cytokine/chemokine magnetic bead panel kits according to the manufacturer’s instructions (EMD Millipore). All assays were performed in duplicate, with the mean value obtained for final analysis. The following cytokines and chemokines were quantified: Interferon gamma (IFNγ), Interleukin 4 (IL-4), Interleukin 5 (IL-5), Interleukin 6 (IL-6), Interferon-inducible protein 10 (IP-10), and Vascular endothelial growth factor (VEGF). Data was quantified on a Luminex MAGPIX instrument (Luminex Corporation) with xPONENT version 4.3 software.We used descriptive statistics to analyze baseline characteristics. Continuous variables are presented as median and interquartile range, while categorical or binary variables are presented as percentages.We used univariate cox regression to determine the association between clinical and sociodemographic predictors with time to KS recurrence. None of the variables satisfied the requirements  to be included in the multivariate model.The Wilcoxon Rank-sum test was used to compare differences in cytokines, chemokines, CD4 counts, and HIV viral loads between groups at baseline and at time of follow-up. The Wilcoxon matched-pairs signed-rank test was used to compare paired data within groups. The Spearman’s rank correlation was used to determine existence of a correlation between CD4 counts and HIV viral loads.We analyzed a total number of 78 samples from a cohort of 39 participants for cytokines, chemokines, CD4 counts, and HIV viral loads. Twenty-seven (27) of these participants were in remission for KS while 12 of them had KS recurrence during the follow-up period . We alsoOf all the clinical and sociodemographic characteristics collected and analyzed, there was none that was significantly associated with KS recurrence .We quantified and compared plasma levels of cytokines and chemokines including IL-4, IL-5, IL-6, IP-10, VEGF-A, and IFNγ at baseline, at the time of follow up and between baseline and follow-up between individuals that had KS recurrence versus those who had sustained remission. These are cytokines and chemokines that have been previously associated with humoral immune response, cell growth and survival, tumor proliferation and survival, chemotaxis, and up- or down-regulation of cancer-related inflammation –13. InteThere was no significant difference in CD4 counts at baseline and at the time of follow-up between individuals who had sustained KS remission and those who had KS recurrence. The HIV viral load was significantly higher at baseline and at the time of follow-up in individuals who had KS recurrence compared to those who had sustained KS remission . There wThis study investigated the factors that are associated with recurrence of HIV-associated KS. Sociodemographic factors including age, sex, smoking status, and alcohol consumption were not associated with KS recurrence. Also, clinical factors including duration of HIV infection, duration of ART uptake, history of multiple KS lesions, history of mucous membrane involvement, and persistent lymphedema were not associated with KS recurrence. These findings suggest that clinical and sociodemographic factors are possibly poor predictors of KS recurrence. However, a larger sample size with additional clinical characteristics, including patients with a history of visceral KS may be required to conclude on these findings.The cytokine IL-5 was significantly higher at the time of follow-up in individuals who had sustained remission compared to those who had KS recurrence. IL-5 is a Th2 cytokine that promotes terminal differentiation of B cells into antibody-secreting plasma cells, and is also an important factor in the differentiation and maturation of eosinophils , 15. In Interleukin-6 was significantly higher at baseline and at the time of follow-up in individuals with sustained remission compared to those who had KS recurrence. IL-6 is a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory effects . In cancThe chemokine IP-10, also known as CXC motif chemokine ligand 10 (CXCL10), was significantly higher at baseline and at the time of follow-up in individuals who had KS recurrence compared to those who had sustained remission. IP-10 is a chemokine that intensifies inflammation by recruiting leukocytes to tissues. IP-10 is associated with HIV infection and disease progression, including the suppression of T cell responses in HIV-infected individuals who are on ART , 23. In A decrease in CD4 counts to significantly low levels below 200 cells/μl is a sign of progression to AIDS in HIV-infected individuals . Low CD4The HIV viral load was significantly higher at baseline and at the time of follow-up in individuals who had KS recurrence compared to those who had sustained remission. Actually, the median HIV viral load was undetectable at both baseline and follow-up in individuals who had sustained remission while it was detectable and slightly increased from baseline levels in those who had KS recurrence. Viral load is a good measure of response to ART, and helps in the monitoring of ART adherence and efficacy . High HIThe small sample size is a limitation when determining the clinical and sociodemographic predictors of KS recurrence. A larger cohort is required to confirm our observations in this regard. In addition, individuals with a history of visceral KS were not included as part of this cohort, as visceral KS may be associated with recurrence. Furthermore, some baseline data from before chemotherapy is also lacking as we only recruited study participants after they went into complete remission. It would be interesting for future studies to compare those in partial remission to those who achieve complete remission. Also, we were unable to determine the trends in the biomarkers over time.For individuals with HIV-associated KS achieving complete remission after chemotherapy, sociodemographic and clinical features are not helpful in predicting disease recurrence. The findings from this study suggest that high IL-5 and IL-6 are associated with sustained remission, while high IP-10 is associated with recurrence of HIV-associated KS. Also, the findings suggest an association between persistently detectable but suppressed HIV viral loads with KS recurrence. A validation of these findings in larger cohorts is needed.S1 File. Dataset(XLSX)Click here for additional data file.


Infectious diseases exacerbated by Antimicrobial Resistance (AMR) are of increasing concern in Sweden, with multi-drug resistant strains associated with new resistance mechanisms that are emerging and spreading worldwide. Existing research has identified that sub-optimal living conditions and poor access to healthcare are significant factors in the spread and incubation of AMR strains. The article considers this linkage and the effort to control the spread of AMR in relation to migrants, highlighting deficiencies in public policy where such individuals are often increasingly exposed to those conditions that exacerbate AMR. In many of the richest countries, those conditions are not accidental, but often direct goals of policies designed with the goal of deterring migrants from staying within host countries. Without engaging with the politics around migration control, the article points to urgent need for more holistic assessment of all public policies that may, however unintentionally, undermine AMR control through worsening living conditions for vulnerable groups. The consequences of prioritizing policies meant to deliberately worsen the living conditions of migrants over avoiding those conditions that accelerate AMR spread, are today made ever apparent where new AMR strains have the potential to dwarf the societal effects of the current Covid-19 pandemic. Infectious diseases exacerbated by Antimicrobial Resistance (AMR) are of increasing concern, with multi-drug resistant strains associated with new resistance mechanisms that are emerging and spreading worldwide. The re-emerging pattern of AMR spread both among humans and animals is of major concern, and its impact in terms of costs for individuals and society. According to the Surveillance atlas of infectious diseases database, AMR is rising in several European countries . In thisForced migration is known to cause physical & psychological stress, due to the circumstances that surround both feeling from their country of origin, and applying for asylum within the host country . MigrantThe newly arrived migrants in Sweden receive information regarding practical issues, including health-care on arrival. They are encouraged to approach the refugee health clinic established in every town; this information is available on the web page “New in Sweden” . Adult aToday, antibiotic therapy is threatened by bacterial resistance that is emerging faster than we can discover and develop new drugs . This isA WHO report on the health of migrants in the Europe states that AMR is increasing worldwide and that forced migration and discriminatory policies in host countries may play a role by worsening living conditions . AlthougAMR is not just a threat for health but carries economic burdens for society. For example, according to an estimate by the Swedish Public Health Agency, by 2050 the number of cases will be four times more than today an cost society approximately 16 billion Swedish Kroner (1.9 billion US dollars) . Much ofIn order to support the newly arrived migrants in their health-care needs on arrival, several measures need to be taken. Many may have succumbed to various medical conditions (incl. exposure to AMR) during transit before arriving in the country, and are at risk of worsening conditions, if not appropriately attended to at the earliest moment. We stress that governing authorities must be able to reassure the newly arrived migrants that health assessments are made immediately and remain confidential with no bearing on their eligibility for the right to stay. Failure to provide such reassurance would risk that those policies intended to protect public health will most likely prove counterproductive by only exacerbating the deteriorating conditions experienced by migrants that favor the incubation of AMR. In addition to health assessments, it is necessary to screen for infectious diseases immediately on arrival and extend prophylactic measures at an earlier stage. If the newly arrived migrants are infected, then this measure could benefit both - the migrants, as well as the host population, by better controlling and preventing the spread of AMR.There are examples of infection control at the time of arrival amongst migrants , 25, butIn addition to health screening to mitigate AMR, we also need to better understand the impact of current migration control policies on the life conditions that may exacerbate the incubation of AMR. Migrant populations are subject to levels of control not experienced by the rest of the population, with likely considerable effects on their vulnerability to AMR. Without reliable baseline data on AMR amongst different populations, it is not possible to directly measure the role migration control policies have on AMR, but there is however substantial data linking those policies to worsening life conditions which are understood as risk factors in AMR , 26–30. There is some evidence that new technologies may help increase access to healthcare for marginalized groups, including migrants. According to a systematic review on health literacy in the eHealth era, mobile apps have a great potential to deliver interactive health services custom-made to people with low health literacy . Such tovia a rhetoric of “national security,” but that in the case of AMR now pose a very real security threat to all national societies unless their negative impact on human lives can be reversed.In conclusion, early access to health-care and health equity among the migrants appear to be critical. If AMR is not tackled within health-care settings, the healthcare risks being ineffective in reaching its goals of good health for all . Whilst The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.ME, P-SM, and SM took the lead in writing this paper and have given an equal contribution to the work with the paper. SK and ZS has read the paper and given comments and suggestions on the writing. All authors have read and approved the last version of the paper.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.


To comparatively evaluate the clinical effectiveness of the single flap approach (SFA) with and without enamel matrix derivative (EMD) in the treatment of supraosseous defects (SDs) associated with deep pockets.n = 10) or in combination with EMD , were retrospectively selected. Clinical parameters  had been assessed at pre-surgery and 12 months post-surgery.Twenty patients, each contributing one SD associated with a deep (≥ 6 mm) pocket and treated with buccal SFA either alone . In SFA+EMD group, 100% of closed pockets was obtained, while 90% of closed pockets was observed in SFA group. Both treatments resulted in a significant CAL gain of 2.1±0.9 mm and 1.9±1.7 mm in SFA and SFA+EMD group, respectively (p= 0.465). In both groups, REC significantly increased 1.0±1.1 mm in SFA group and 1.1±1.1 mm in SFA+EMD group (p= 0.722).Complete wound closure was observed in 70% and 80% of defects treated with SFA and SFA+EMD, respectively. Treatments resulted in a significant PD reduction of 3.1±1.0 mm (Within their limits, the findings of present study suggest that SFA may represent a valuable option for the surgical treatment of SDs associated with deep pockets. EMD did not result in a significant clinical benefit to the procedure.SFA may represent a valuable option in obtaining pocket closure when treating SDs associated with deep residual pockets. Among periodontitis-related lesions, supraosseous defects (SDs) are characterized by the coronal location of the gingival sulcus/periodontal pocket with respect to the bone crest . SDs areSDs have been indicated as the least predictable periodontal lesion when a regenerative procedure is performed  and stilIn 2007, the single flap approach (SFA) was proposed as a simplified surgical procedure for the regenerative treatment of intraosseous defects , 16. TheBased on the hypothesis that EMD may enhance the clinical outcomes of SFA surgery at SDs, the present study was performed to comparatively evaluate the clinical effectiveness of SFA with and without EMD in the treatment of SDs associated with a deep periodontal pocket.The present study is a retrospective analysis of consecutively treated cases. Patients were selected among those seeking care at the Research Centre for the Study of Periodontal and Peri-implant Diseases, University of Ferrara, Italy, and one private dental office in Ferrara, Italy. All the clinical procedures were performed in accordance with the Declaration of Helsinki and the Good Clinical Practice Guidelines (GCPs). Each patient signed an informed consent to surgical treatment. The study protocol (reference code: 1096-2020-Oss-UniFe) was approved by the local Ethical Committee .Clinical Procedures for details) between January 2013 and February 2018, a patient contributing a SD treated with SFA alone was selected by matching either the highest value of pre-surgical PD  or the distance  between the cementum-enamel junction and the interproximal bone crest (CEJ-BC).Data were retrieved from the record charts of periodontal patients treated at the Research Center for the Study of Periodontal and Peri-implant Diseases  and a private dental office in Ferrara. For each patient contributing a SD treated with SFA+EMD  periodontal conditions compatible with a diagnosis of stage III–IV periodontitis , 29, as SDs were considered eligible for analysis when presenting the following characteristics: (i) CEJ-BC≥ 4 mm at the tooth aspects facing the defect; (ii) depth of the intrabony component ≤2 mm ; (iii) residual PD≥ 6 mm and persisting bleeding on probing (BoP) following treatment phase I–II [All surgical procedures were performed by two experienced periodontal surgeons  by using ×2.5–3.5 magnifying loupes. Minimal modifications to the incision design of the SFA were adopted , 16. BriSutures were removed 2 weeks after surgery. The patients were asked to abstain from mechanical oral hygiene procedures in the surgical area for 4 weeks. A 0.12% chlorhexidine mouthrinse (10 mL twice daily for 6 weeks) was used to support local plaque control. Each patient was enrolled in a monthly recall program for the first 3 months and was reviewed according to personal needs thereafter. Each session included reinforcement of oral hygiene procedures and supragingival plaque removal.The following patient-related parameters related to the time of the surgical procedure were extracted from the record charts: age (in years), gender, and smoking status (non-smoker/current smoker).Immediately before surgery and 12 months after surgery, the following measurements were performed by using a periodontal probe with 1-mm increments : PD, measured from the gingival margin to the bottom of the pocket; BoP, recorded as positive or negative if bleeding was detected during or after PD assessment; CAL, measured from the CEJ  to the bottom of the sulcus/pocket; and interdental gingival recession (REC), measured from the CEJ  to the gingival margin at the interproximal aspect . PD, BoP, CAL, and REC had been evaluated at the mesio-buccal and mesio-palatal/lingual sites of the tooth distal to the SD and at the disto-buccal and disto-palatal/lingual sites of the tooth mesial to the SD. For each defect, only the site with highest pre-surgery PD among the two mesial sites and two distal sites was considered for the present analysis.The distance between the CEJ and the interproximal bone crest (CEJ-BC), as assessed immediately after the completion of the surgical debridement in mm at the site with highest PD, was also extracted from the patient record charts.On digital photographs taken at 2 weeks post-surgery, wound healing at the incision level was evaluated according the Early Healing Index (EHI) . EHI is closed pockets) was the primary outcome.Data were entered into a statistical software . Each patient contributed the study with one SD. If a patient presented two or more eligible SDs, one SD was randomly selected for the present study. The proportion of SDs with a 12-month PD≤4mm  was calculated as the percentage ratio between CAL gain and pre-surgery CAL. Data were expressed as mean ± standard deviation.χ2 test or Fisher’s exact test. Inter-group comparisons were performed with Mann-Whitney U test and χ2 test or Fisher’s exact test. The level of significance was set at 5% for all statistical tests.Within-group comparisons were performed with Wilcoxon test for paired observations and n= 10). Due to the presence of a 100% value in one of the two groups, however, the calculation failed, thus making impossible to generate the post hoc statistical power of the study.Among previous studies investigating the adjunctive effect of EMD in the treatment of SDs, only the study by Iorio-Siciliano et al.  reportedTwenty patients , each contributing with one SD, were included. Thirteen defects had been treated  at the Research Center for the Study of Periodontal and Peri-implant Diseases, University of Ferrara, Ferrara, and 7 had been treated  in a private dental office in Ferrara. Patient and defect characteristics are reported in Table p= 0.959).Patient distribution according to EHI within each treatment group is reported in Table Clinical measurements as well as their 12-month changes are reported in Table p= 0.770). In SFA+EMD group, 100% of closed pockets was obtained, while 90% of closed pockets was observed in SFA group. These results were parallel by a significant reduction in the number of BoP positive sites in both groups (p< 0.001). Treatments resulted in a significant CAL gain of 2.1±0.9 mm (SFA group) and 1.9±1.7 mm (SFA+EMD group) (p= 0.465). rCAL gain amounted to 28.5 ±13.1% and 22.9 ±18.4% for SFA and SFA+EMD, respectively (p= 0.304).A significant PD reduction of 3.1±1.0 mm was observed in both groups, resulting in a mean 12-month PD of 3.5 mm in either SFA + EMD or SFA group (p= 0.722).Both treatments resulted in a similar, significant increase in REC, which amounted to 1.1±1.1mm for SFA+EMD and 1.0±1.1 mm for SFA (The presence of deep ≥6 mm), bleeding pockets after phase I and II of periodontal therapy represent a strong indication to implement periodontal therapy with additional treatments, including surgical options [ mm, bleeIn the present investigation, the SFA, which was proposed for the treatment of intraosseous defects , has beeIn our material, SFA plus EMD resulted in a CAL gain of 1.9±1.7 mm, with no significant difference with SFA group. Differently, previous studies showed that the adjunctive use of EMD significantly enhanced the outcomes of access flap when the latter was performed according to papilla preservation techniques, with a CAL gain ranging from 2.8±0.8  to 3.4±0Several studies have reported beneficial clinical effects on early soft tissue healing following the use of EMD –33. In tThe results of the present study must be considered with respect to some limitations. The latter consist mainly of the retrospective nature of the study, the lack of preliminary data to calculate the sample size a priori and the impossibility to generate the post-hoc statistical power of the study. Moreover, the lack of complementary information from radiographic exams has precluded a further analysis on radiographic bone level changes after treatment.In conclusion, the results of the present study indicate that (i) SFA represents a valuable option in the treatment of SDs associated with presenting deep pockets and (ii) SFA+EMD do not show in a significant added benefit to the procedure in terms of CAL gain.


However, the size of microdialysis probes and sampling rate may limit this technique’s ability to provide an accurate assessment of drug effects in microneural environments. A novel electrochemical method known as multiple-cyclic square wave voltammetry (M-CSWV), was recently developed to measure second-to-second changes in tonic dopamine levels at microelectrodes, providing spatiotemporal resolution superior to microdialysis. Here, we utilized M-CSWV and fast-scan cyclic voltammetry (FSCV) to measure changes in tonic or phasic dopamine release in the nucleus accumbens core (NAcc) after acute cocaine administration. Carbon-fiber microelectrodes (CFM) and stimulating electrodes were implanted into the NAcc and medial forebrain bundle (MFB) of urethane anesthetized (1.5 g/kg i.p.) Sprague-Dawley rats, respectively. Using FSCV, depths of each electrode were optimized by determining maximal MFB electrical stimulation-evoked phasic dopamine release. Changes in phasic responses were measured after a single dose of intravenous saline or cocaine hydrochloride . In a separate group, changes in tonic dopamine levels were measured using M-CSWV after intravenous saline and after cocaine hydrochloride . Both the phasic and tonic dopamine responses in the NAcc were augmented by the injection of cocaine compared to saline control. The phasic and tonic levels changed by approximately x2.4 and x1.9, respectively. These increases were largely consistent with previous studies using FSCV and microdialysis. However, the minimal disruption/disturbance of neuronal tissue by the CFM may explain why the baseline tonic dopamine values (134 ± 32 nM) measured by M-CSWV were found to be 10-fold higher when compared to conventional microdialysis. In this study, we demonstrated phasic dopamine dynamics in the NAcc with acute cocaine administration. M-CSWV was able to record rapid changes in tonic levels of dopamine, which cannot be achieved with other current voltammetric techniques. Taken together, M-CSWV has the potential to provide an unprecedented level of physiologic insight into dopamine signaling, both in vitro and in vivo, which will significantly enhance our understanding of neurochemical mechanisms underlying psychiatric conditions.For over 40 years,  Substance dependence is a global public health problem. A 2019 survey revealed 20.4 million people aged 12 or older in the United States suffered from substance use disorder . Around in vivo is a major challenge.Dopamine is widely implicated in addiction. Its functions include determining the incentive value of naturally occurring positive rewarding stimuli  . PreviouThere are generally two distinct patterns of spike firing exhibited by neuronal dopamine-containing cells in the mammalian midbrain: tonic activity and phasic burst activity . Phasic Tonic concentrations of dopamine in the brain have been typically quantified in the sub-nM range using microdialysis . MicrodiIn contrast, electrochemical methods, such as fast-scan cyclic voltammetry (FSCV), have features that are well-suited to quantitatively measure changes in extracellular dopamine concentrations . FSCV prFor the measurement of tonic dopamine levels in the brain in real time, several electrochemical techniques were developed such as fast-scan controlled-adsorption voltammetry (FSCAV) , square In this study, we aim to elucidate the acute effects of cocaine administration on phasic dopamine release by using FSCV and tonic dopamine levels by using M-CSWV in the nucleus accumbens core (NAcc). Cocaine is one of the most common illicit drugs with an increasing prevalence of use and dependence .Nine male Sprague-Dawley rats  were used for this study. Rats were kept in social housing in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited vivarium following a standard 12-h light/dark cycle at constant temperature (21°C) and humidity (45%) with ad libitum food and water. The present studies were approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic, Rochester, MN. The NIH Guide for the Care and Use of Laboratory Animals guidelines  were followed for all aspects of animal care.in vivo biofouling.CFMs were fabricated using an established standardized CFM design at Mayo Clinic. . BrieflyEach rat was anesthetized with urethane  and administered buprenorphine  for analgesia. Following anesthesia, they were placed in a stereotaxic frame . Respiratory rate , hind-paw and tail pinch were used to monitor the physiological state and depth of anesthesia. Using a standard rat atlas , three tThe stimulating electrode in the MFB and CFM in the NAcc were first adjusted to obtain a robust stimulation-evoked dopamine signal via FSCV . MFB was chosen as it is known to induce dopamine release in the NAcc . For theFor the tonic dopamine group, the system was switched to the M-CSWV sensing technique with electrodes at the same position after identification of an optimal CFM position in the NAcc using FSCV. Cocaine at the same dose and route as above was administered after baseline and post-saline recording. Further recordings were performed for another 90 min to monitor the potentially lasting effects of cocaine on tonic dopamine levels. Dynamic background subtraction and capacitive background current modeling was used to eliminate large capacitive background currents, allowing tonic dopamine concentrations to be measured every 10 s . BecauseAfter experimentation, changes in dopamine release for phasic studies were determined by calibration of CFMs using a flow cell injection apparatus; whereas for tonic dopamine levels, calibration with dopamine solutions were used . The medin vivo data compared to previous models such as the diffusion gap model. We applied the restricted diffusion model to our data to extract best-fit estimates for the parameters Rp, kR, kU, and kT . Significance was set at p < 0.05. In the phasic experiments, three planned paired t-tests were performed . In the tonic experiments, the peak level after cocaine injection was compared to baseline and saline levels.Statistical analysis was performed using ratio two-tailed paired t-test, p = 0.0124, n = 4 rats), baseline  and 60 min after cocaine administration . With repeated stimulation performed for 60 min after injection, the peak level dropped to the pre-injection baseline level (Cocaine administration consistently led to enhancement of stimulation-evoked dopamine responses . The evone level .p = 0.028)  . Cocainet-test, p = 0.002, n = 5 rats)  .in vivo. By utilizing the M-CSWV technique with its unrivaled temporal resolution and high spatial resolution provided by CFMs, we have demonstrated a more precise picture of how tonic dopamine dynamics change in response to acute cocaine administration.This is the first study to characterize changes in tonic dopamine levels in the NAcc in the presence of cocaine in near real-time with an electrochemical technique U was lowered after cocaine administration, which is expected since cocaine is a competitive antagonist of the dopamine transporter and kU is directly proportional to reuptake rate   . Also, ihibitor) . This su cocaine . After sWhile our technique focuses on a single analyte, dopamine, and therefore may not be as comprehensive as microdialysis studies, precise measurement of dopamine alone is still highly important. Dopamine has a major role in the reward circuit and is one of the key neurotransmitters in the pathophysiology of addiction . Newer tAnother notable characteristic of this study is that the animal experiments were performed under anesthesia in an acute setting using a single dose of cocaine. While we appreciate that addiction is often secondary to chronic drug use in humans, acute experiments offer insight into the first step of the pathophysiological process. Additionally, this study paves the way for future chronic experiments by proving the feasibility of this technique to study the effects of other drugs of abuse. In the future, it is our intention to apply the M-CSWV intraoperatively, particularly in the context of neurological  and psychiatric  disorders.Although cocaine is known to enhance dopamine transmission in the nucleus accumbens, this is the first study that utilized M-CSWV to measure accumbal tonic dopamine levels, and to characterize the effect of cocaine on these levels in near real-time. Overall, this technique provides unprecedented insight into the temporal changes in dopamine dynamics, and it will likely be of much value in future addiction studies.Supplementary video. An example of tonic dopamine measurements obtained from the NAcc in a single rat with saline then cocaine i.v. injections. Upper panel shows the color plot and the lower panel shows the changes in tonic dopamine concentration over time, where the black line denotes the stabilization period, blue line denotes control  and red line denotes post-cocaine measurements.