High‐throughput DNA analyses in cancers allow the detection of molecular abnormalities that can guide patients' treatment. The collection of a tumour DNA is mainly performed on a biopsy, an invasive procedure for the patients. Another less invasive method consists in using a fine needle. We showed that fine‐needle biopsies are as suitable as a classical biopsy for DNA analysis. High‐throughput molecular profiling of solid tumours using core needle biopsies (CNB) allows the identification of actionable molecular alterations, with around 70% success rate. Although several studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to the sensitivity of the less invasive fine‐needle aspiration (FNA) compared to CNB to detect molecular alterations. We aimed to prospectively evaluate the potential of FNA to detect such alterations in various tumour types as compared to CNB in cancer patients included in the SHIVA02 trial. An in‐house amplicon‐based targeted sequencing panel (Illumina TSCA 99.3 kb panel covering 87 genes) was used to identify pathogenic variants and gene copy number variations (CNV) in concomitant CNB and FNA samples obtained from 61 patients enrolled in the SHIVA02 trial (NCT03084757). The main tumour types analysed were breast (38%), colon (15%), pancreas (11%), followed by cervix and stomach (7% each). We report 123 molecular alterations  among which 98 (80%) were concordant between CNB and FNA. The remaining discordances were mainly related to deletions status, yet undetected alterations were not exclusively specific to FNA. Comparative analysis of molecular alterations in CNB and FNA showed high concordance in terms of variants as well as CNVs identified. We conclude FNA could therefore be used in routine diagnostics workflow and clinical trials for tumour molecular profiling with the advantages of being minimally invasive and preserve tissue material needed for diagnostic, prognostic or theranostic purposes. CNBcore needle biopsyCNVcopy number variationCTcomputerized tomographyEDTAethylenediaminetetraacetic acidFDRfalse detection rateFNAfine‐needle aspirationGGaugeNGSnext‐generation sequencingQCquality controlRPMIRoswell Park Memorial InstituteSNVsingle nucleotide variationTSCATruseq Custom Amplicon1With the large deployment of next‐generation sequencing (NGS) and the increasing knowledge on cancer genomics goes an accelerated demand for molecular analyses.Precision medicine clinical trials in oncology, based on molecular analyses to prevent, diagnose and treat patients, use image‐guided biopsies as the gold standard starting material to perform these analyses , 2, 3. HEGFR mutations in 90% of the cases  and FNA [147.8 ng·µL−1 (2.9–414 ng·µL−1)]. The large majority of CNB and FNA samples had a DNA concentration > 100 ng·µL−1 ] in FNA samples. The mean percentage of mapped reads was equal to 81.78% (1.46–98.63) for CNB and 83.52% for FNA (12.60–98.37). Finally, when considering a depth ≥ 100X , the coverage was of 93.11% (3.88–98.29) for CNB and 95.43% (76.53–98.22) for FNA ] in CNB samples NA Table .r = 0.831 and r = 0.815 respectively).No statistical difference was found between CNB and FNA samples regarding DNA concentrations, DNA ratios (both 260/280 and 260/230), reads number, percentage of mapped reads, percentage of mapped reads on target, and percentage of depth ≥ 100X. The percentage of mapped reads and the depth of coverage were both significantly correlated and similar between CNB and FNA samples (3.3n = 43) were amplifications and 83% (n = 217) were deletions . Among the CNV, 17% , 49% (21/43) of amplifications and 51% (110/217) of deletions were detected in both CNB and FNA Fig. . RemarkaA high correlation between CNB and FNA regarding pathogenic variants and CNV alterations was observed. For pathogenic variants, the correlation of allelic frequencies was 0.743; for CNV alterations, 90% of pairs of FNA and CNB showed a significant correlation of copy number profiles at false detection rate (FDR) 5% Fig. .n = 23 amplifications and n = 15 homozygous deletions) were subsequently retained as clinically relevant , followed by PIK3CA , KRAS, and ESR1  and CDKN2A/CDKN2B  , 85 pathogenic variants and 38 CNV alterations  Table . Interess) Table .3.4versus 13% (16/123) of discordant actionable molecular targets  of all validated alterations. Among them, 76% (53/70) were concordant between CNB and FNA 23 of allts Table . Discord4Biopsies are the gold standard material to establish a diagnosis and to perform molecular profiling in precision oncology , 32, 33.In the current study, we prospectively analysed the relevant molecular alterations detected in concomitant CNB and FNA sample pairs obtained from a series of patients enrolled in the SHIVA02 trial, using an in‐house NGS‐based gene panel.versus cytospot surface for FNA). However, it is important to note that for our molecular analysis, cellularity did not affect the quality and quantity of DNA needed, or the quality of targeted NGS required in our study.First, by comparing QCs related to CNB and FNA sample types, we observed that both DNA  and NGS QCs  showed no difference between CNB and FNA, underlying their equivalent suitability for molecular assays. We noticed a difference regarding tumour cell content between CNB and FNA samples, which could be explained by the difference in references used for the evaluation of tumour cell percentage  and in a second step of validation of the Total data according to an algorithm .The bioinformatics analysis  showed a significant correlation between CNB and FNA regarding both the detection of pathogenic variants and CNV alterations. Seventy‐three per cent of pathogenic variants, 49% of amplifications and 51% of deletions were commonly detected in corresponding CNB and FNA pairs. Remarkably, after validation and selection of driver molecular alterations , the rate of common alterations detected between CNB and FNA from the same pair considerably increased up to 81% of pathogenic variants, 91% of amplifications and 53% of deletions. For pathogenic variants, this rate is in accordance with previous studies , 19, 24.Altogether, these observations underlie the equal reliability of both sample types for the identification of clinically relevant molecular alterations in tumour cells.In this study, only metastasis samples were analysed. In fact, for theranostic purposes and in order to include patients with metastatic tumours in early trials to give matched targeted therapy, molecular analyses on metastasis seem more relevant. However, it could be interesting to perform the same study on primary tumours as they might have a higher variability of clones than in the metastasis, the latest originating from one major clone under treatment selection. Indeed, two samples (CNB and FNA) taken from the primitive tumour could harbour a higher heterogeneity and show more molecular discordances compared to the metastasis , 38.Four patients out of 61 did not harbour any relevant validated molecular alteration . For these cases, sampling procedures were successful, except for patient 45 for which the biopsied lesion appeared to be a nontumoral lymph node rather than a metastasis (which was lately confirmed by PET scan). Histologies were validated by the pathologists, and no anomaly was reported during NGS process. The absence of validated molecular alterations in these samples could therefore either be explained by the absence of alterations at the DNA level in these tumours, or by a lack of coverage of the altered genes in the targeted panel used.CDKN2A only detected in the FNA, while a heterozygous deletion of CDKN2A was found in the CNB. This result still led to orientation to a treatment by CDK4‐6 inhibitor  in the frame of SHIVA‐02, but unfortunately, the patient died one month later. Patient 27 harboured an homozygous deletion of PTEN in FNA sample only but not in the CNB. This result did not affect the decision taken within the SHIVA02 molecular board , as PTEN inactivation is not part of the treatment algorithm. Yet, this new result led to a discussion in the local Molecular Tumour Board (MTB) for eventual treatment by mTOR inhibitor [Two patients had targetable alterations detected only in FNA: patient 19 and patient 27. Patient 19 had a homozygous deletion of nhibitor .These discordances between molecular results from CNB and FNA could be explained by intratumoral heterogeneity , 38, 40,TP53, PIK3CA, KRAS, ESR1 and CDKN2A/B were the most frequently altered genes, which is in accordance with the tumour types screened, as the majority were metastatic breast, colon and pancreas cancers [Among validated alterations across all the samples,  cancers , 42. Reg cancers , 44. The5Our results show that: (a) both CNB and FNA samples are suitable for molecular analysis, with the same DNA and NGS qualities; (b) relevant validated molecular alterations are equally detected in both CNB and FNA samples from the same patient, with a higher consistency after than before validation of data; and (c) actionable targets were identified in both CNB and FNA. We demonstrated the feasibility of real‐time molecular screening of biomarkers from FNA samples and incorporation into clinical workflows to orientate diagnosis or therapeutic decisions. Finally, our study also emphasizes the critical role of data analysis and validation of relevant molecular alterations in order to have reliable, comparable data between different sample types.With the increasing need to identify tumour‐specific molecular alterations to improve diagnosis and treatment algorithms for personalized therapy, FNA samples can be of critical utility, especially for patients with nonbiopsiable lesions or advanced diseases where small specimen is the only material available, or, when performed in parallel to CNB, molecular profiling on FNA could allow to save tissue material for other purposes. This could open the doors of NGS analyses and new therapeutic options to more patients.IB, VS, JM‐P, MK and CLT conceived and designed the study. JM‐P, EG, PG, VS and MK developed the methodology. JM‐P, MK, SA, CF, OM, EG, PG, PR, EB, DB, FR, SH, M‐PS, PT, AM, DL, MF, AV‐S and CG involved in data acquisition. JM‐P, EG, PG and CD analysed and interpreted the data. CD, JM‐P, MK, CG, IB and CLT wrote, reviewed and revised the manuscript. IB, CLT, AV‐S, JM‐P, CD, MK, CG, EG, PG, CC, SA, CF, OM and CL gave administrative, technical or material support . IB, VS, CLT and MK supervised the study.CLT has participated in advisory boards from MSD, BMS, Merck Serono, GSK, Amgen, Novartis, Roche, Rakuten, Seattle Genetics, Nanobiotix and Astra Zeneca.Fig. S1. Representation of sample acquisition in cancer patients. Concomitant FNA and CNB are performed during the same procedure. A 22 Gauge needle is inserted into the guide needle to perform the first sampling (1) which is the FNA. Then, an 18 or 16 Gauge cutting needle is inserted into the guide needle to perform the CNB. The second sampling corresponding to CNB is performed close from the first FNA sampling, either by going deeper into the tissue with the needle (2) or changing the needle's angle (2′).Click here for additional data file.Fig. S2. Percentages of tumour cells in CNB and FNA samples determined by pathologists. Data are presented as mean ± SEM. Mann–Whitney test was used to compare the group of CNB samples versus FNA samples.Click here for additional data file.Fig. S3. Repartition of DNA concentration ranges in CNB and FNA samples analysed. Data are presented as mean ± SD. Kruskal–Wallis test followed by Dunn's test was used to compare CNB and FNA samples DNA concentrations within the 3 groups  and no significance was found.Click here for additional data file.Fig. S4. Number of pathogenic variants detected in CNB and FNA per patient.Click here for additional data file.Table S1. Cancer type, tumour cell percentage, and DNA and NGS libraries quality control metrics of CNB and FNA samples.Table S2. Description of validated molecular alterations in CNB and FNA samples of 61 patients from the SHIVA02 trial.Click here for additional data file.


The composites nanofibers were exposed under 5, 30, and 100 kGy of irradiation dose. The electron-beam irradiation showed no morphological damage to the fibers, and slight reduction in the water-contact angle and mechanical strength at the higher-irradiation doses. The chain scission was found to be a dominant effect; the higher doses of electron-beam irradiation thus increased the in vitro degradation rate of the composite nanofibers. The chemical interaction due to irradiation was indicated by the Fourier transform infrared (FTIR) spectrum and thermal behavior was investigated by a differential scanning calorimeter (DSC). The results showed that the electron-beam-induced poly L-lactide acid/carboxy-methyl starch/β-tricalcium phosphate (PLLA/CMS/β-TCP) composite nanofibers may have great potential for bone-tissue engineering.Nanofibrous materials produced by electrospinning processes have potential advantages in tissue engineering because of their biocompatibility, biodegradability, biomimetic architecture, and excellent mechanical properties. The aim of the current work is to study the influence of the electron beam on the poly L-lactide acid/ carboxy-methyl starch/β-tricalcium phosphate (PLLA/CMS/β-TCP) composite nanofibers for potential applications as bone-tissue scaffolds. The composite nanofibers were prepared by electrospinning in the combination of 5%  Nanofibers have acquired huge interest in the past few decades due their great properties that can be used in a wide range of applications. Nanofibers can provide great surface area over volume, high connected pores, and good mechanical properties ,2,3,4. DIn tissue engineering of nanofibers, cell attachment and proliferation is the most important aspect that needs to be considered when developing new scaffold material . BesidesThe effect of ionizing radiation on PLLA has been reported by several authors, either by gamma or electron beam ,27,28. CIn this work, we used an electron beam to study the properties of composite natural–synthetic nanofibers. The incorporation was a natural polymer of carboxy-methyl starch (CMS) derived from a sago starch with PLLA. The CMS provided the carbohydrate source, as present in the extra-cellular matrixes (ECM). CMS consists of amylose and amylopectin in its molecular structure with a 1–4 glycosidic bond that can enhance cell and material interaction. β-TCP particles were used to initiate the potential of this composite nanofiber to be used in bone-tissue engineering. The presence of β-TCP particles in the nanofibers would enhance the initiating mechanism of the cell interaction. β-TCP particles have a rough surface morphology that encourages attachment to bone cells. This study is an extension of our previous work in composite nanofibers. The previous study was carried out to determine the ratio between PLLA/CMS and PLLA/CMS/β-TCP to investigate the effect of the mix ratio of all components . The phyIn tissue engineering, control of the degradation rate is one of the critical issues for synchronization of degradation kinetics to the rate of natural tissue formation. Control of the degradation rate is a challenging task. The novelty of this work is that electron-beam-induced PLLA/CMS/β-TCP composite nanofibers have been used to enhance the in vitro degradation of the PLLA matrix as an eco-friendly green method. The electron-beam irradiation doses and degradation rate-dependent effects on composite nanofibers have been examined.l-lactide acid) with the inherent viscosity of 2.32 dl/g was obtained from BioInvigor . β-TCP nanopowder was purchased from Berkeley Advanced Biomaterials Inc., USA, with an average particle size of approximately 250 nm. Carboxy-methyl starch was prepared from local sago starch. 10 g of sago starch was stirred in 300 mL isopropanol  with an addition to 30 wt.% NaOH in a reactor flask equipped with a reflux condenser and burette. In this work, the carboxymethylation process of sago starch was prepared as described by Bohari et al. [l-lactide acid) solutions with 7 wt% mol concentration were prepared by dissolving the granule PLLA in dichloromethane by stirring the mixture for 24 h using a magnetic stirrer. The CMS solution was prepared at 10 wt% concentration and mix with PLLA in 5 % v/v of CMS content in PLLA. 0.25% of β-TCP powders were mixed in PLLA/CMS solution in the presence of sodium dodecyl sulfate (0.2 wt%). The samples were ultrasonic and stirred for 72 h and rotary-mixed to homogenize the mixture.Poly. The syringe pump  was placed vertically with a flow rate of 0.006 mL/min, and the nanofiber mat was collected on aluminum foil.The surface of the fibers was characterized using a scanning electron microscope . The images were collected at ×2000 and ×10, 000 magnification at 10 kV. The fiber diameter was analyzed and calculated by using ImageJ  and Stack Graphic 4.0 software .–1, which was performed at 16 scans per sample.Chemical interaction analysis was performed by using Fourier transform infrared (FTIR) spectroscopy . The FTIR spectrum was measured in the spectral range of 400 to 4000 cm–1 to avoid oxidation during heating.Changes in thermal properties were investigated by using a differential scanning calorimeter . About 2.35 mg samples were heated from 20 °C to 250 °C at a heating rate of 10 °C /min. Nitrogen gas was purged to the sample approximately 35 mL minThe crystalline and amorphous regions of samples were investigated using an XRD instrument . Samples were scanned from 5° to 60° of 2θ angle with a step size of 0.02°/sec. The X-ray source was from the CuKα with 1.5140 of the wavelength. Xpert High Score Plus software  was used to analysis of the XRD.Wettability of the nanofiber surfaces was determined by water-contact angle . A 2 μL of distilled water was dropped on the surface of the nanofibers. 130 WCA data points were collected in 12 s.2) with 10 mm of the gauge length for the tensile test. The samples were removed from the aluminum foil using a paper frame with double-sided tape attached to it. The frame provided additional support to the sample for handling during the testing process. Tensile testing was conducted using a 20 N load cell  equipped with a micro-stepper motor system  and an OMRON RXRX25 data logger to record the load. The elongation was determined by 1.0 mW Omron laser detector with detection limit of 2.5 ms/600 nm. The tensile test was conducted at 0.5 mm min–1 of the crosshead velocity. The tensile strength was taken as the maximum stress of the stress–strain curve.The composite PLLA/CMS/β-TCP nanofibers were punched into a rectangular shape  with 5 kGy per pass to reduce the heat generated from the electron beam. Voltage and current were set at 2 MeV and 1 mA, respectively. The distance between energy source and samples were set at 2 cm to optimize the electron alignment when hitting the samples.After irradiation, samples were immersed in a PBS medium for degradation study. The medium was refreshed every three days to provide fresh ions to the samples. Samples were incubated in the oven at 36.7 °C for 7, 14, 30, and 60 days. After 7, 14, 30, and 60 days, samples were removed and rinsed with deionized water before drying for 96 h at 45 °C. The weight of the samples was taken before and after immersion to obtain the percentage of mass loss after incubation.The surface hydrophilic and hydrophobic behaviors of composite nanofibers were analyzed by water-contact angle measurement (WCA). The change in irradiation doses exhibited a small change in WCA. WCA reduced about 6 % from 125° to 118° after a 100 kGy irradiation dose . The pos–1 has increased, indicating the amount of OH group formed from the degradation of the polymer chain in the CMS molecular structure  was also observed at 100 kGy at 1735 cm–1 of the absorption band , melting temperature (Tm), and cold crystallization temperature (Tcc) was determined by the DSC thermogram. The thermogram exhibited two endothermic peaks and one exothermic peak  during the electrospinning process, which were α and β, with a different melting temperature [The thermal behavior of the composite nanofibers was investigated by DSC analysis. The glass transition temperature  at both amorphous and crystalline regions. At higher doses, degradation may occur by hydrogen breakage due to the higher oxygen permeability into the amorphous region . Alkyl fTensile strength was found to decrease with the increase of irradiation doses. Tensile strength reduced from 8.43 MPa for non-irradiated to 3.21 MPa after irradiation at 100 kGy . This coInvestigation of in vitro degradation was carried out by immersing the composite nanofibers into PBS solution for 7, 14, 30, and 60 days. Changes in morphology and the mass loss indicated the degradation of composite nanofibers. the fiber diameter of composite nanofibers changed slightly after beam irradiation.WCA dropped from 125 to 118° due to the formation of hydroxyl groups after the highest dose of beam irradiation.FTIR, DSC, and XRD results indicated that there are chemical changes to the structure of composite nanofibers after the highest dose of beam irradiation.A tensile test was run to observe the mechanical strength before and after beam irradiation. Apparently, the material decreased its mechanical properties by 2.6 times due to the breaking of the polymer chains after the highest beam-irradiation dose. Moreover, rapid reduction in the elongation showed that polymer degradation took place after beam irradiation.SEM images after electron-beam irradiation showed that degradation took place after 30 and 60 days in vitro test.Electron-beam irradiation enhanced the in vitro degradation of PLLA matrix since the PLLA component has a longer degradation period. However, higher-irradiation doses retarded the mechanical integrity of the composite.Electron-beam-induced composite PLLA/CMS/β-TCP nanofiber mats have been prepared in order to analyze the beam effect at various doses  on the physical and chemical morphology and degradation behavior of materials. SEM and WCA were used to evaluate surface morphology while FTIR, DSC, and XRD were used for the chemical morphology of the composite nanofibers. Furthermore, tensile and in vitro degradation tests were run to evaluate the degradation of samples under various beam intensities. The results can be concluded as follows:This method to prepare degradable nanofibers is helping to develop and design new biomaterials. Electron-beam-irradiated composite PLLA/CMS/β-TCP nanofiber mats showed rapid biodegradation demonstrating their potential for tissue-engineering applications, specifically bone tissue.


Three-component molecular brushes with a polyimide backbone and amphiphilic block copolymer side chains with different contents of the “inner” hydrophilic (poly(methacrylic acid)) and “outer” hydrophobic (poly(methyl methacrylate)) blocks were synthesized and characterized by molecular hydrodynamics and optics methods in solutions of chloroform, dimethylformamide, tetrahydrofuran and ethanol. The peculiarity of the studied polymers is the amphiphilic structure of the grafted chains. The molar masses of the molecular brushes were determined by static and dynamic light scattering in chloroform in which polymers form molecularly disperse solutions. Spontaneous self-assembly of macromolecules was detected in dimethylformamide, tetrahydrofuran and ethanol. The aggregates size depended on the thermodynamic quality of the solvent as well as on the macromolecular architectural parameters. In dimethylformamide and tetrahydrofuran, the distribution of hydrodynamic radii of aggregates was bimodal, while in ethanol, it was unimodal. Moreover, in ethanol, an increase in the poly(methyl methacrylate) content caused a decrease in the hydrodynamic radius of aggregates. A significant difference in the nature of the blocks included in the brushes determines the selectivity of the used solvents, since their thermodynamic quality with respect to the blocks is different. The macromolecules of the studied graft copolymers tend to self-organization in selective solvents with formation of a core–shell structure with an insoluble solvophobic core surrounded by the solvophilic shell of side chains. Modern methods of polymer synthesis provide great opportunities to obtain macromolecules with a complex architecture such as molecular brushes, star-shaped polymers, hyperbranched polymers or dendrimers, that is, systems containing blocks or components of different chemical nature ,2,3,4,5.Among the polymers with a complex architecture, molecular brushes received a significant amount of attention due to their shape and unique properties ,14,15,16Macromolecular brushes with different nature of the backbone and side chains are of special interest because they may adopt a wide variety of conformations in selective solvents ,18,19. TMulticomponent molecular brushes with amphiphilic block copolymer side chains are promising objects. In fact, such systems are ternary block copolymers of complex architecture, which combine three blocks differing in their chemical structure. Such diphilic graft copolymers possess a unique complex of properties due to the ability to form ordered micellar structures by the self-organization of macromolecules in selective solvents . The selb-poly(methyl methacrylate) (PMAA-b-PMMA) were synthesized by the ATRP method [Previously, molecular brushes with a hydrophobic polyimide (PI) backbone and amphiphilic diblock copolymer side chains of poly(methacrylic acid)-P method . The patP method .This work is a continuation of previous research and aims to systematically study the behavior of molecular brushes with a polyimide backbone and amphiphilic block copolymer side chains in selective solvents and to establish the effect of the architecture and structural parameters of macromolecules on their conformational characteristics and self-assembly behavior. One of the important tasks is to analyze the influence of the ratio of hydrophilic and hydrophobic blocks in the side chains on the self-organization of three-component brushes in solutions.tert-butyl methacrylate) (PtBMA) followed by chain extension of methyl methacrylate (MMA) from living chain ends of PI-g-PtBMA. Amphiphilic molecular brushes with hydrophilic blocks of poly(methacrylic acid) (PMAA) in side chains were obtained by selective acidic hydrolysis of PtBMA blocks in side chains of the prepolymer. A description of the materials used and their purity are described in The synthesis of the investigated copolymers and their characterization were described in detail previously . The tartert-butyl methacrylate and methyl methacrylate) was characterized by gas chromatography using a Shimadzu GC-2010 Plus gas chromatograph equipped with a flame ionization detector and an Agilent J&W DB-WAX GC Capillary Column . 1H NMR spectra of polymerization products were recorded using a Bruker AC200 (200.1 MHz) spectrometer with DMSO-d6 or CDCl3 as solvents.In order to obtain molecular brushes, samples of the multicenter polyimide macroinitiator with 2-bromoisobutyrate initiating groups in each repeat unit were used. The conversion of monomers (tert-butyl methacrylate) or poly(methacrylic acid) and outer poly(methyl methacrylate) blocks are given in The structures of molecular brushes with a hydrophobic polyimide backbone and block copolymer side chains consisting of inner poly(b-PMMA) side chains were cleaved from the backbone using the selective alkaline hydrolysis described earlier in [In order to determine the reliable structural parameters of the studied copolymers, the homo- (PtBMA) and block copolymer (PtBMA-rlier in . Molar mrlier in .g-(PMAA-b-PMMA), hereafter will be labeled as N* and correspond to the numbers of the precursor samples PI-g-(PtBMA-b-PMMA) in The samples of molecular brushes with amphiphilic block copolymer side chains, PI-As is known, the behavior of amphiphilic polymers in solutions strongly depends on the thermodynamic quality of the solvent with respect to the components. Therefore, the choice of solvents for research is an important task. As can be seen from g-PtBMA, PI-g-(PtBMA-b-PMMA) and PI-g-(PMAA-b-PMMA)) were molecularly dissolved in chloroform, while aggregates were present in dimethylformamide (DMF), tetrahydrofuran (THF) and ethanol. Therefore, their molar mass characteristics were determined in chloroform. The molar masses of the PI macroinitiator were measured in DMF, which is a thermodynamically good solvent for PI.The synthesized molecular brushes  and dynamic light scattering (DLS) using a Photocor Complex instrument . The light source was the Photocor-DL diode laser with the wavelength λ = 659.1 nm and controllable power up to 30 mW. The correlation function of the scattered light intensity was obtained using the Photocor-PC2 correlator with 288 channels and processed using the DynalS software. Toluene was used as a calibration liquid, whose absolute scattering intensity Rh were obtained by the DLS method. In the solvents in which the aggregation occurs, we could determine the radii of gyration Rg and hydrodynamic radius Rh in those cases when the distribution of the intensity of the scattered light by the particle size was unimodal. Under multimodal distribution, the Rh value was estimated. Experimental error under determination Rh is 10%In chloroform, the asymmetry of the scattered light was absent for all polymers, even for high-molar mass polymer brushes, therefore, the radii of gyration of scattering objects could not be determined, and their molar masses were obtained by the Debye method. The values of the hydrodynamic radii Prior to the experiments, the scattering cells were rinsed with benzene, evacuated for 15 min and filled with dust-free air. The solutions were prepared at room temperature. All solutions were stored for at least 12 h prior to measurements, in order to ensure a complete equilibration. The investigations were performed in dilute solutions at 21 °C. All solutions were filtered twice into dust-free cells using Chromafil polyamide filters  with the pore size of 0.45 µm.Atomic force microscopy (AFM)  in tapping mode using commercial tips  with a resonance frequency of 300 kHz and a spring constant of ~50 N/m was utilized to characterize the morphology of the molecular brushes. The samples for AFM measurements were prepared by spin casting a chloroform solution of c = 0.1 mg/mL in concentration on a mica surface at 2000 rpm for 1 min. The measurements were carried out in air at room temperature and constant humidity of 55%.b-PMMA were synthesized through formation of an intermediate molecular brush with PtBMA-b-PMMA side chains followed by selective hydrolysis of PtBMA chains. This research has focused on three-component molecular brushes with two types of side chains: precursor PtBMA-b-PMMA and target PMAA-b-PMMA. As described previously in the experimental part, all blocks of the studied molecular brushes  have different solubility in solvents of different nature. Molecular and conformational characteristics were determined for precursor brushes PI-g-(PtBMA-b-PMMA) using hydrodynamic methods. The self-assembly behavior in selective solvents was investigated for the target molecular brushes with amphiphilic block copolymer side chains PI-g-(PMAA-b-PMMA). This approach implies strict consistency between the molar masses of the precursor and amphiphilic side chains. Namely, while preserving the polymerization degree of side chains of molecular brushes, the molar mass of the “inner” block of the side chains of the target brushes should decrease by a factor of ~1.7 compared to the side chains of the precursor brushes due to the hydrolysis of the tert-butyl methacrylate units. To prove the validity of this approach, two-component brushes PI-g-PtBMA and PI-g-PMAA were specially synthesized. The efficiency of the hydrolysis reaction was confirmed by 1H NMR spectroscopy. As shown in g-PtBMA precursor brushes were analyzed by SEC. To estimate the chain length of the molecular brushes with poly(methacrylic acid) side chains after selective acidic hydrolysis of precursor molecular brushes, the single macromolecules of PI-g-PMAA were visualized using tapping-mode AFM  and PI-g-(PMAA-b-PMMA) in chloroform. There was no Rh dependence on concentration for most of the samples , and sample* PI-g-(PMAA-b-PMMA)  chains, the calculated weight-average molar mass of PI-g-PtBMA is from ~500,000 to ~650,000 g·mol−1, which agrees well with the value determined by the light scattering method  with block copolymer side chains, increases the weight-average molar mass of the copolymer by about 380,000 g·mol−1. Hence, the calculated molar mass of PI-g-(PtBMA-b-PMMA) is from 880,000 to 1,030,000 g·mol−1, agreeing reasonably with the experimentally determined value for this sample, Mw = 870,000 g·mol−1  and PI-g-(PMAA-b-PMMA) samples is approximately 190,000 g·mol−1, and, accordingly, the calculated molar mass of the target copolymer is from 690,000 to 840,000 g·mol−1. The experimental value of molar mass of PI-g-(PMAA-b-PMMA) is equal to 690,000 g·mol−1  nm. The obtained value of the hydrodynamic radius Rh-D for the PI macroinitiator corresponds with good accuracy to a dependence of the type of Mark–Kuhn–Hauwink–Sakurada equation used for the hydrodynamic radius [The conformational analysis for the PI macroinitiator  showed tc radius .Rh-D slightly increases (Rh-D for PI-g-PMMA and PI-g-PS [Rh-D for PI-g-PMMA и PI-g-PS in selective solvents (PI-g-PMMA and PI-g-PS are graft copolymers with a PI backbone and poly(methyl methacrylate) and polystyrene (PS) side chains, respectively) [g-PMMA and PI-g-PS in selective solvents (heptanon-3 and cyclohexane-chloroform mixed solvent in the volume ratio of 99:1), in which a collapsed PI backbone is shielded from the solvent by side chains [Unfortunately, due to the small asymmetry of the scattered light, it was impossible to determine the radius of gyration of the studied brushes. Therefore, the conformation of their molecules was estimated by analyzing the values of the hydrodynamic radii of macromolecules. In the transition to graft copolymers, ncreases , being m PI-g-PS  in thermctively) . Therefoe chains . Thus, mg-(PMAA-b-PMMA) were dissolved non-molecularly . This makes it possible to assume that the species responsible for the fast mode are the isolated macromolecules. The observed decrease in molecules size in DMF and THF in comparison with Rh-D in chloroform can be explained by the worst thermodynamic quality of these solvents and the reduction in macromolecular size caused by compression of the backbone and side chains. The radius of slow-mode particles decreased with dilution, and extrapolation to zero concentration was used to determine the hydrodynamic radii Rh-s of these species. The particles with radius Rh-s were aggregates which are formed due to the interactions of the PI backbone and/or PMAA blocks in side chains. Note that the aggregates in DMF are larger than in THF probably due to the worst thermodynamic quality of the latter. The Rh-s values are on average an order of magnitude higher than the radius Rh-D. Comparing the Rh-D and Rh-s values, one can estimate the aggregation degree az. Within the framework of the rigid sphere model, az = (Rh-s/Rh-D)3 . The obtained az values lie in the range from 1000 to 3000, while no dependence on the molar mass of the sample and the solvent nature was found.In other solvents, the target copolymers PI-ecularly . In DMF ntration . Their ag-(PMAA-b-PMMA), a unimodal distribution of light scattering intensity on the hydrodynamic radius of scattering particles was observed  in ethanol, the ratio Rg/Rh-m of the gyration radius Rg to the hydrodynamic radius was lower than 1.1 (In ethanol solutions of PI-observed . As can than 1.1 . This mag-(PMAA-b-PMMA), in particular the content of PMMA blocks, strongly influenced the aggregate characteristics in ethanol solutions. The decrease in the PMMA fraction in side chains led to a decrease in aggregate size. This fact is illustrated, for example, by a comparison of the Rh-m and Rg obtained for samples 3* and 1*, as well as samples 5* and 6* (g-(PMAA-b-PMMA) differed in length of the outer PMMA block that leads to a change in the Rh-m value. In addition, an increase in the y/x ratio caused a decrease in the Rh-m radius. Accordingly, aggregates in solutions of sample 3* have the smallest dimensions (The composition of molecular brushes PI-* and 6* . With simensions .g-(PMAA-b-PMMA) differed by structural parameters, namely the grafting density of the side chains, their length and the ratio of hydrophilic and hydrophobic segments in the side chains. The structure and composition of synthesized molecular brushes were confirmed by comparing molar masses of copolymers and their components.Series of amphiphilic molecular brushes with a polyimide backbone and amphiphilic block copolymer side chains were synthesized by ATRP. Poly(methacrylic acid) was the inner hydrophilic block and poly(methyl methacrylate) was the outer hydrophobic block. The target samples of PI-g-(PMAA-b-PMMA) samples was determined by their structural parameters and nature of the solvent. In molecularly disperse solutions (in chloroform), the molecules of the synthesized graft copolymers had compact sizes and resembled core–shell structures, the core of which was a collapsed PI backbone and the shell was formed by side chains. Aggregation of macromolecules was observed in most solvents. The aggregate size depended on the thermodynamic quality of the solvent in relation to the copolymer components, as well as on the parameters of the macromolecular architecture. Particularly, in ethanol, an increase in the PMMA content led to a reduction in the aggregate size and an elongation of the outer PMMA block caused a decrease in the hydrodynamic radius. In this solvent, aggregates had a sufficiently symmetrical shape, close to spherical.Solution behavior of PI-


Cardiac computed tomography angiography (CCTA) is widely used as a diagnostic tool for evaluation of coronary artery disease (CAD). Despite the excellent capability to rule-out CAD, CCTA may overestimate the degree of stenosis; furthermore, CCTA analysis can be time consuming, often requiring advanced postprocessing techniques. In consideration of the most recent ESC guidelines on CAD management, which will likely increase CCTA volume over the next years, new tools are necessary to shorten reporting time and improve the accuracy for the detection of ischemia-inducing coronary lesions. The application of artificial intelligence (AI) may provide a helpful tool in CCTA, improving the evaluation and quantification of coronary stenosis, plaque characterization, and assessment of myocardial ischemia. Furthermore, in comparison with existing risk scores, machine-learning algorithms can better predict the outcome utilizing both imaging findings and clinical parameters. Medical AI is moving from the research field to daily clinical practice, and with the increasing number of CCTA examinations, AI will be extensively utilized in cardiac imaging. This review is aimed at illustrating the state of the art in AI-based CCTA applications and future clinical scenarios. Coronary computed tomography angiography (CCTA) represents an excellent tool for the evaluation of patients with suspected stable coronary artery disease (CAD) –6. ThereCCTA represents an important step in clinical management of patients with suspected CAD; however, it is important to keep in mind that the majority of CCTA results in no evidence of significant CAD , 13. FurThe application of artificial intelligence (AI) in cardiac radiology is aimed at facilitating the management of patients with suspected CAD ranging from diagnosis to prognostic stratification . In partThe AI industry has seen massive growth in a variety of fields in the past decade, with the field of medicine not being an exception. The basis of AI is mathematics and computer science with the three main pillars being (1) big data, (2) high performance computing infrastructure, and (3) algorithm development. The exponential growth in digital storage capabilities, data collection systems, and computing power enabled AI applications in a wide variety of fields. The current digital era leads to an increased amount of information, which is beneficial to the development of AI algorithms. The technological developments make it possible to develop algorithms that are able to deal with the large amount of data and complexity typical of the digital era we live in.With AI currently entering the medical field, early stage applications have mainly focused on automatization of medical tasks; more recently, the focus has shifted towards prognostication and risk prediction. Many studies investigate the potential role of AI in supporting clinicians in their day-to-day tasks, assisting in workflow optimization, quantification, diagnosis and prognostication, and reporting. However, many clinical AI applications are currently only used in a research setting and are far from being implemented into clinical practice. There are examples of successful AI implementation . The DatThe European commission released a white paper on AI in February 2020, including statements on the use of AI for medical purposes , 28. TheBesides the legal framework for medical AI, there are some ethical considerations that will play a key role . With thThe grading and coronary segments involved with obstructive of CAD have been associated with a worse prognosis .Often assessment of CAD stenosis is time consuming, requiring multiplanar reconstruction selection of the best phase in the cardiac cycle for a correct assessment of coronary arteries and depends on the experience of the reader . After tZreik et al. developed a 3D CNN that was able to characterize the plaque and evaluate the grading of stenosis . The autKang et al. developed an AI technique based on a two-step algorithm with a vector machine that was useful for the evaluation of CAD stenosis . On a poYoneyama et al. evaluated the possibility to identify the grading of coronary stenosis and its impact in terms of ischemia using a cohort of patients who underwent CCTA and perfusion single photon emission computed tomography (SPECT) . The autVan Hamersvelt et al. developed an algorithm of AI that evaluated the presence of significant CAD using a combined approach of AI that analyzes the myocardium and compared it with invasive FFR . They foTwo studies developed an automated approach of CADRADS in clinical practice , 39.Muscogiuri et al. evaluated the impact of a new deep learning algorithm based on CNN for the classification of CAD-RADS in a cohort of 288 patients who underwent CCTA for a clinical indication . The timAnother important application of automatic CAD-RADS classification was shown by Huang et al. . The autCoronary Artery Calcium Score (CACS) is an independent predictor of adverse cardiovascular events –42.CT images for the evaluation of calcium score are often acquired using an ECG-gated, no contrast technique and segmented calculating a calcium volume, and mass obtaining a specific value of calcium score . CurrentThe evaluation of CACS using an AI algorithm can definitely speed up the time of reporting.One of the first articles describing the evaluation of CACS using an algorithm of AI was developed by Isgum et al. . The autSandsted et al. evaluated the performance of an AI algorithm for the evaluation of CACS compared to semiautomated CACS . The autk of 0.85 for Agatston risk score between the automated and reference scores [Despite CACS traditionally being evaluated using ECG-gated scans, recently, Takx et al. analyzed the impact of AI for evaluation of CACS in non-ECG-gated and noncontrast images acquired in a cohort of patients undergoing a CT for lung cancer screening . In a coe scores ; howevere scores .The combination of CACS analysis and lung cancer screening can be a powerful combination in clinical practice to identify patients that may benefit from therapy.Wolterink et al. described the application of an automated algorithm for the evaluation of CAC in 250 patients who underwent CCTA . The autFinally, Van Velzen et al. evaluated calcium scores from different CT without contrast . 7240 exAssessment of plaque composition is extremely important in CCTA reporting; indeed, identification of fibrous or calcified plaques can be extremely important for patient management . PresencThe application of AI can facilitate and speed up the analysis of CCTA providing accurate information on plaque analysis in a relative short time.Zreik et al. developed an algorithm that was able to identify the plaque morphology and severity of stenosis . From a r: 0.88) and noncalcified plaque (r: 0.98) with a good correlation compared to manual segmentation [Another application of AI for identification of different plaque types was developed by Dey et al. The authors developed an algorithm that automatically differentiated calcified plaque  for the evaluation of plaque characteristics has been demonstrated to characterize plaque . Using rKolossvary et al. evaluated the radiomic features of plaques showing napkin ring sign (NRS) which has been associated with poor outcome . The autAn ML approach can identify the presence of thin cap fibroatheroma (TCFA) overcoming the technical limitation of CCTA . In partRecent research and development in AI has been applied in multiple potential applications of cardiac CT-derived myocardial ischemia assessments. Most software applications herby deal with CT-derived fractional flow reserve (FFR) for the detection of hemodynamically significant CAD. Only few studies of AI applications using CT perfusion have been published so far. In terms of CT-FFR, ML solutions have been provided by only one vendor , 57. Howp < 0.05). In accordance with the results of the MACHINE registry, several single-center studies have evaluated the diagnostic performance of ML-based CT-FFR, reporting sensitivities and specificities ranging from 79% to 82% and 91% to 94%, respectively [p = 0.04). Another substudy of the MACHINE registry assessed the impact of gender on the diagnostic accuracy of ML CT-FFR with no significant difference in the AUCs in men when compared to that of women  [ML-based CT-FFR employs a multilayer neural network framework that was trained and validated offline against the former CFD approach by using a virtual dataset of 12.000 synthetic 3D coronary models . The cliectively , 60. ML-ectively  investig = 0.89) . OverallOnly few studies have assessed the use of AI for CT perfusion. However, CT perfusion offers a field with great potential for the application of AI especially for automated identification of perfusion defects and myocardial segmentation. Preliminary results have demonstrated an AUC of 0.73 by using different ML approaches for automated segmentation and delineation of the left ventricle when compared to manual segmentation by an expert reader . In anotFocusing on outcome, there are several manuscripts that show the impact of CAD depicted on CCTA and prognosis , 65. An Motwani et al. evaluated the impact of an ML algorithm for prognostic stratification in a large cohort of 10030 patients with follow-up of 5 years and an endpoint of mortality . A totalVan Rosendael et al. developed a model for risk stratification based on a population from the CONFIRM registry . The priTesche et al., in a small cohort of patients, developed an AI algorithm for risk stratification in patients who underwent CCTA with follow-up of 5.4 years . The autIn CCTA, the role of AI may be important for further radiation dose reduction  without Human interpretation, despite their experience, is still prone to fatigue. Furthermore, the time of training of expert readers requires years of experience. The application of AI in CCTA will not substitute the cardiac radiologist; rather, AI will represent a helpful tool for reporting and prognostic stratification. Indeed, following the ESC guidelines , over thFurthermore, CCTA analysis is moving toward a model of precision medicine. The analysis of coronary stenosis grading is not sufficient alone. A comprehensive CCTA report needs to provide information regarding characterization of plaque and its hemodynamical effect; furthermore, the joint evaluation of clinical parameters can be helpful to stratify the patients in terms of worse outcome and can be helpful for individual treatment plans.It is plausible that an algorithm will be composed for automatic analysis of CCTA images followed by detection of myocardial ischemia . SubsequStrict legislation focused on the application of AI in cardiac imaging will be necessary to clarify the medico-legal aspects of the AI algorithm application. Furthermore, the development of an AI algorithm implies the analysis of a large amount of data; this aspect is extremely important if we consider the legal aspects due to privacy.All these aspects need to be clarified in the future before we consider the application of AI in routine clinical practice.In the future, AI will be integrated in the CCTA workflow. AI applications will greatly benefit CCTA practice reducing the reporting time and providing a more accurate quantitative-based approach to CAD management, moving the entire field in the direction of precision-based medicine. However, before we can widely implement AI solutions in our clinical practice, we need to carefully validate the algorithms in the light of standards for good medical practice and new medical device utilization and carefully address possible issues on data protection, legal framework, and ethical principles.


We measured in vitro binding of 2-DPMP to rat striatum and accumbens DAT by means of quantitative autoradiography with a selective DAT-radioligand [125I]RTI-121. We evaluated the effects of intravenously administered 2-DPMP on extracellular dopamine in the accumbens-shell and striatum using in vivo microdialysis in freely moving rats. We used dynamic modeling to investigate the interactions of 2-DPMP within DAT, in comparison with cocaine and amphetamine. 2-DPMP potently displaced the radioligand in the accumbens and striatum showing dose-dependence from 0.3 to 30 μM. IC50 values were: 5.65 × 10-7M for accumbens shell and 6.21 × 10-7M for dorsal striatum. Dose-dependent responses were also observed in accumbens-shell and striatum in vivo, with significant increases in extracellular dopamine levels. Molecular dynamics simulations identified contrasting conformational changes of DAT for inhibitors (cocaine) and releasers (amphetamine). 2-DPMP led to molecular rearrangements toward an outward-facing DAT conformation that suggested a cocaine-type effect. The present combination of molecular modeling with experimental neurobiological procedures allows for extensive characterization of the mechanisms of drug actions at DAT as the main molecular target of stimulants, and provides an insight into the role of dopamine in the molecular and neurobiological mechanisms of brain responses to stimulant NPS that have addictive potential. Such knowledge reveals the risk of addiction related to NPS use. The research presented here can be adapted for other psychostimulants that act at their membrane protein targets.Stimulant drugs, including novel psychoactive substances  have addictive potential which their users may not realize. Stimulants increase extracellular dopamine levels in the brain, including the reward and addiction pathways, through interacting with dopamine transporter (DAT). This work aimed to assess the molecular and atomistic mechanisms of stimulant NPS actions at DAT, which translate into biological outcomes such as dopamine release in the brain’s reward pathway. We applied combined  Typical drugs of addiction, stimulants such as cocaine or amphetamine, have been known to share among them the ability to activate the brain’s reward system and increase extracellular levels of dopamine (DA) in the mesolimbic pathway, and preferentially in the nucleus accumbens (NAc) . ElevateStimulants can share similar structural moieties, such as phenylethylamine which is a common structural feature found embedded in many stimulants like amphetamine and methylamphetamine and is also found in the naturally occurring neurotransmitter dopamine , while ain vitro methods, and in silico, using molecular modeling of interactions between the drugs and DAT (in vivo evidence of dopamine release caused by those stimulants.Stimulants can be found among novel/new psychoactive substances (NPS) ; it is i and DAT . We havein vitro assays in human embryonic kidney 293 cells (HEK 293) that express DAT, NET and SERT, 2-DPMP was most potent at DAT: the IC50 values were 0.07 µM for DAT, 0.14 µM for NET and >10 for SERT  . In the for SERT . 2-DPMP for SERT . Importafor SERT .in vitro as well as in vivo approaches, the latter testing the effects on DA release in the brain’s reward pathway in freely moving rats. We hypothesized that those in vitro and in vivo findings can be interpreted at the atomistic level, by means of in silico methods of computational biophysics with molecular modeling and simulations, and based on the consensus that there are distinct differences in the mode of action between dopamine releasers/substrates and dopamine reuptake inhibitors. Regardless of whether the binding and transport of these compounds are driven by entropy (substrates and releasers) or enthalpy (inhibitors)   they indibitors) .As the pharmacology of 2-DPMP has been reasonably described , that toin vitro autoradiography study, 8-week-old male Wistar rats  were used. They were kept six per cage on a 12/12-h light/dark cycle (lights on at 7 AM) with food and water freely available. Temperature and humidity were 18°C–22°C and 55% ± 15% respectively. Rats were treated in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and sacrificed by cervical dislocation.For the in vivo microdialysis study, adult male Wistar rats  were housed in groups of four per cage under an inverted 12:12-h light/dark cycle and at a constant temperature of 22°C ± 2°C and humidity of about 60%. Tap water and standard food were available ad libitum in the home cage. The in vivo animal experiments were conducted in the University of Cagliari  and carried out in accordance with the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research according to Italian (D.L. 116/92 and 152/06) and European Council directives (609/86 and 63/2010) and in compliance with the approved animal policies by the Ethical Committee for Animal Experiments  and the Italian Ministry of Health (Aut. N.162/2016-PR). Every effort was made to avoid pain and suffering, and to reduce the number of animals used.For the 125I]RTI-121 (specific activity 81.4TBq/mmol) was purchased from Perkin Elmer.All chemicals, including desoxypipradrol hydrochloride solution D-082, were purchased from Sigma Chemicals . The radioligand for the dopamine transporter, RTI-121 in the NaPB with increasing concentrations of 2-DPMP (0–30 µM) for 60 min at room temperature. Nonspecific binding was assessed in the presence of 200 µM nomifensine (control – “block”). Kodak BioMax MR films were applied over the rinsed and air-dried slides for three days. Autoradiograms were analyzed using MCID™, Version 7.0, Imaging Research Inc. . Flat-field correction was applied. Each drug concentration was tested in six brains. The dorsal striatal  and accumbens (NAc) regions of interest (ROIs) were sampled in duplicates for relative optical density, left and right ROI values were averaged and their means were calculated to assess the specific binding.Brains were rapidly removed and frozen at −40°C, then stored at −80°C. Frozen brains were cut into 20-µm coronal sections to harvest the ventral and dorsal striatum areas at +1.7mm to −0.3mm against bregma . Serial 2  methanol, pH 5; the detector settings were +125 mV (first electrode/oxidation) and −175 mV (second electrode/reduction). Extracellular dopamine was monitored for a total period of 180 min.Rats were anesthetised with isoflurane gas (4%–5%), and maintained under anesthesia using a breathing tube under a scavenging system while placed in a stereotaxic apparatus, and implanted with a catheter consisting of a polyethylene tubing  in the right jugular vein and stable fixed in the mid-scapular region of the back. During the same surgical session, rats were implanted with vertical dialysis probes aimed at the NAc shell and CPu. The following coordinates were used according to 2 . Samplespost mortem. High reproducibility of the procedure allowed reducing the group size with the intention of limiting the animal use to minimum.At the end of the experiment, all animals were sacrificed under the anesthesia, the probes were gently removed, and the brains harvested and cut in coronal sections with a vibratome to check the correctness of a microdialysis probe location in every brain. Fiber placement was determined as consistent with the coordinates by 125 I]RTI-121 was analyzed in the presence of increasing concentrations of the drugs in both CPu and NAc shell.Autoradiography data were analyzed using two-way ANOVAs followed by post-hoc Tukey’s test. Data were expressed as mean percentage ± standard error of mean (SEM) against the control value. Binding of RTI-121 was examined in NAc and CPu. 125I]RTI-121 in the presence of varying concentrations of 2-DPMP, ranging from 0  to 30 µM. 2-DPMP caused a marked, concentration-dependent reduction in the radioligand signal in the brain tissue, indicating potent competition between the drug and the radioligand in NAc shell and CPu. IC50 values were: 5.65 × 10-7M for NAc shell and 6.21 × 10-7M for CPu.The displacement of , dose , time  and a significant brain area × time , dose × time , and brain area × dose × time interaction . In the NAc shell, in silico part of the study aimed to investigate the molecular changes when 2-DPMP is bound to DAT and compare it to the DA releaser, amphetamine that induces an inward-facing conformation of DAT and the DAT inhibitor, cocaine that locks DAT in an outward-facing conformation.The + and Cl- obtained positions for all three drugs near a common high affinity binding site that is near the primary substrate-binding S1 site  vestibule  , the NPSCocaine, on the other hand, adopts a position in the S1 site where there is minor destabilization of the EC gate Y156–F320 bond at these time scales. As the extracellular salt-bridge R85-D476 remains closed, we interpret it as showing that the DAT is maintained in the original outward-facing conformation  in the IC vestibule  the primary binding site S1 is the same for all three investigated drugs, (2) the mechanisms of interaction of DAT with amphetamine, cocaine and 2-DPMP highlight the critical role of the intracellular and extracellular salt-bridges, R60-D436 and R85-D476 respectively’ and negatively charged aspartate residue (D79) in stabilizing the drug in the S1 binding site, and (3) MD simulations show contrasting conformational changes of DAT for inhibitors and releasers.To sum up, our in vitro, in vivo, and in silico approaches to assess the dopaminergic effects of 2-DPMP as the chosen NPS stimulant, selected on the basis of the existing literature , depending on the dose and route of administration . The mosvia DAT like a substrate, stimulates the efflux of intracellular DA; MD simulations have shown structural rearrangements of the transporter toward an inward-facing conformation, while cocaine binding inhibits DA transport through forcing DAT to remain in the outward-facing conformation  and serotonin transporter (SERT) contribute to the stimulant characteristics to a varying degree across the range of stimulant drugs . Both noin vivo microdialysis experiments would have provided information on the level of behavioral stimulation caused by the drug. Additionally, the time scales for the simulations in this study may not be enough to sample the conformational space accessed by these compounds in DAT. Further studies can be expected to expand on these time scales, with an emphasis on comparing the signature dynamics of various drugs of addiction and stimulations at DAT. These observations will include structural changes of important interhelical distances of transmembrane TM1b–TM10 and TM6a–TM10 and TM1a–TM6b, representative of EC vestibule openings and IC vestibule openings, respectively.The present study is not free from limitations. Thus, quantitative behavioral observations of rat mobility, freezing, piloerection, grooming, sniffing, gnawing, rearing and scratching during the in silico and in vitro methods. The research presented here and in our previous studies  and European Council directives (609/86 and 63/2010) and in compliance with the approved animal policies by the Ethical Committee for Animal Experiments  and the Italian Ministry of Health (Aut. N.162/2016-PR).BL, MS, and JO-J were responsible for the study concept and design. BL and JO-J conducted the ligand binding experiments and analyzed the data. ML facilitated and designed the microdialysis study. BL collected and interpreted the microdialysis data with ML’s input. HS parameterized the ligands and performed the docking studies. MS performed the molecular simulations and interpreted the findings. JO-J, BL, and MS drafted the manuscript. All authors critically reviewed and edited the content and approved the final version.This project was supported in part by grants of the European Commission . Further financial support was provided by the EU Commission‐targeted call on cross-border law enforcement cooperation in the field of drug trafficking—DG Justice/DG Migrations and Home Affairs (JUST/2013/ISEC/ DRUGS/AG/6429) Project EPS/NPS , and by the Drug Policies Department, Presidency of the Council of Ministers, Italy (project: “Effects of NPS: development of a multicentre research for the information enhancement of the Early Warning System” to ML), and RAS-FSC 2014-2020 to ML .http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/L000253/1).The following computational resources are gratefully acknowledged: ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.


Mikania micrantha is a noxious invasive plant causing enormous economic losses and ecological damage. Soil microbiome plays an important role in the invasion process of M. micrantha, while little is known about its rhizosphere microbiome composition and function. In this study, we identified the distinct rhizosphere microbial communities of M. micrantha, by comparing them with those of two coexisting native plants (Polygonum chinense and Paederia scandens) and the bulk soils, using metagenomics data from field sampling and pot experiment. As a result, the enrichment of phosphorus-solubilizing bacteria Pseudomonas and Enterobacter was consistent with the increased soil available phosphorus in M. micrantha rhizosphere. Furthermore, the pathogens of Fusarium oxysporum and Ralstonia solanacearum and pathogenic genes of type III secretion system (T3SS) were observed to be less abundant in M. micrantha rhizosphere, which might be attributed to the enrichment of biocontrol bacteria Catenulispora, Pseudomonas, and Candidatus Entotheonella and polyketide synthase (PKS) genes involved in synthesizing antibiotics and polyketides to inhibit pathogens. These findings collectively suggested that the enrichment of microbes involved in nutrient acquisition and pathogen suppression in the rhizosphere of M. micrantha largely enhances its adaptation and invasion to various environments. The rhizosphere is the interface where the complex interactions among soil, microbes, and the host plant are maintained . Plants Alnus trabeculosa increased the soil bacterial diversity in the invaded regions  turnover rates in soil  could reduce the richness and abundance of native species in the invaded regions, or even dramatically change the local ecological system . Compare regions . Anotherlability . In addilability . C. macu natives . An inva natives . For exa natives . Solidag in soil , and C.  in soil . Invasiv in soil .Mikania micrantha (Asteraceae family), an extremely fast-growing vine, is one of the top 100 worst IAS in the world  were replicated six times (two plants per pot with 7 kg fresh soil) and put in a greenhouse.In order to test the contribution of rhizosphere bacteria to icrantha . The seeM. micrantha is an ecologically harmful weed in the natural environment. We chose the natural field of M. micrantha monoculture with the dominant two coexisting native plants (P. chinense and P. scandens) in the dry riverbed of Liuxi River in Guangzhou City. We separated five (5 m by 5 m) sampling plots by more than 200 m and used the same method of pot experiment to collect 15 rhizosphere samples of three plants and five control samples, which is in the uninvaded area near M. micrantha monoculture by more than 500 m, for a total of 20 samples.Three months later, we randomly selected five replicates of each treatment and the rhizosphere soil of three plants and control soil were collected. Plants were removed carefully and shaken lightly; then, the soil remaining attached to the root surface was collected with sterile water. The separated soil solution was centrifuged at 8000 r/min for 10 min to collect soil samples. The collected soils were stored at −80°C until use for microbial community analysis. A combination of bacteria cell lysis steps was applied before DNA extraction. The soil microbial cells were subjected to six freeze–thaw cycles . Next, DNA was extracted from all samples using the PowerSoil DNA isolation kit following the manufacturer’s protocol . The DNA quality and quantity were checked by the Nanodrop and Qubit device, and the DNA quantity of each sample was at least 1 μg. Then, DNA fragments (200–400 bp) were processed by ultrasonic instrument . The library was constructed using TruSeq DNA PCR-Free Library Prep Kit as per standard protocol  and then sequencing was performed on Illumina HiSeq 2500 with each sample having about 10 Gb sequencing data.1, resulting in a clean and high-quality reads data with average error rate < 0.001. Then, the clean reads from each sample and pooled for four groups  were assembled by Megahit (v1.1.3). After filtering the contig length less than 500 bp, gene prediction was performed using MetaProdigal (v2.6.3), and then we filtered out the gene models with cds length less than 102 bp. The protein models of each sample and each group were also performed using MetaProdigal (v2.6.3). The non-redundant gene catalog was obtained using the genes predicted from each sample and each group by cd-hit-est (v4.6.6) with the criteria of identity > 95%, and overlap > 90% (parameter “−c 0.95 −n 10 −G 0 −aS 0.9”). The non-redundant protein catalog was obtained from the combination of protein files of each sample and each group by in-house software fishInWinter.pl2.The raw reads were cleaned by removing adaptor sequences, low-quality sequences, host sequences, and unpaired reads by in-house software clean_adapter, clean_lowqual, and filter_unpaired_reads.plE value < 1e-5) against the KEGG protein database (release 79) using DIAMOND software  software with the parameter “blastp –evalue 10 –max-target-seqs 250” . CARMA3 software . To calcsoftware . The alisoftware . Based oThe bacteria and genes involved in soil microbial P-solubilizing and mineralization, pathogen, and defense were searched based on previous publications and are shown in At the gene level, Shannon index was used to analyze microbial alpha diversity using the non-redundant genes of individual samples. The overall differences in the bacterial community structures were calculated by non-metric multidimensional scaling (NMDS) using non-redundant genes of individual samples based on Bray–Curtis dissimilarity values and implemented in in R package “Phyloseq.”M. micrantha, P. chinense, and P. scandens were determined using Kruskal–Wallis test with Dunn’s multiple comparison (BH methods for multiple tests adjustment). The relative abundance of microbial species and functional genes involved in P solubilization, pathogens, and defense in the control soil and rhizospheres of three plants is compared using Kruskal–Wallis test with Dunn’s multiple comparison (BH methods for multiple tests adjustment).Based on the relative abundance profiles at the phyla or genera level, the significantly differential abundances in the control soil and rhizospheres of M. micrantha genome project, deposited in NCBI (SRR8936416–SRR8936475) and AGIS ftp-site3, we investigated the microbial structure of the rhizosphere of M. micrantha and two native plants (P. chinense and P. scandens). The microbial alpha diversity (Shannon index) at the gene level showed no significant difference between the control soil (bulk soil) and rhizospheres (P > 0.05)  in the rhizospheres (Using the metagenomic data and non-redundant gene set of  > 0.05) . Howeverded site . Moreoveded site . The domoroflexi , which woroflexi . The comospheres . This suM. micrantha and the two native plants were analyzed at the genus level, at both pot experiment and invaded site. From the metagenomic data, genes could be classified to the genus level by CARMA3 software. The relative abundance of genus in each sample was calculated according to reads count at the genus level. In total, the top 69 genera (relative abundance > 0.01%) accounted for 94.8% of the total relative abundance of classified genera, and 45 of them were enriched  in rhizospheres compared to the bulk soil, most of which belong to Proteobacteria and Actinobacteria , is an essential element for plant growth and development , playinge plants .Pseudomonas, Bacillus, Enterobacter, and Burkholderia , which might also contribute to the solubilization of soil unavailable phosphorus. Taken together, the recruitment of these PSM would help to increase the available P content in M. micrantha rhizosphere.Phosphorus solubilizing microorganisms (PSMs), such as holderia , can incscandens ; howeverectively . Similarectively . In the s putida  and Enteasburiae  were morzosphere . On the M. micrantha and two native plants (P < 0.05) (M. micrantha (P = 0.045) and P. chinense (P = 0.07), whereas phoA showed no significant difference (P > 0.05) (P > 0.05)  . The gen > 0.05) . The rel > 0.05) . These rThe plant-associated microbiome, as the second genome of the plant, has great influence on plant growth and health . To suppFusarium oxysporum  and P. scandens (sixfold and fourfold) rhizosphere compared to M. micrantha rhizosphere (P. scandens (P = 0.04) than in M. micrantha rhizosphere   , whereasM. micrantha and two native plants (P. chinense and P. scandens) at invaded field site and pot experiment. Since the pot experiment lasted only 3 months, and each plant grew independently without competition, obvious microbial differences between pot experiment and invaded site were observed  and P. chinense (P = 0.02)   in the iF. oxysporum and R. solanacearum, as well as T3SS, were depleted in M. micrantha rhizosphere compared to the native plants  have allelopathic effects on native plants and antibacterial activities (M. micrantha rhizosphere. In summary, the fewer pathogens and more protective microorganisms that inhabit the M. micrantha rhizosphere potentially benefit root growth and nutrient uptake, thus possibly enabling the successful invasion. However, there is a lack of difference in the soil microbes in M. micrantha between the origin and invaded one. Evidences for the resource availability and pathogen release in soil of invasive plants would require combined tests in the native and invaded ranges. Hence, in order to comprehensively understand the role of soil microorganisms in M. micrantha invasion, the metagenome of M. micrantha rhizosphere in the native range and the differences to their introduced range would need to be studied in the future. Although we showed the differences of microbial community and functional genes among the rhizosphere of three plants, the observed changes would require further experimental study.Invasive plants may benefit from introduction to new regions where they can escape pathogens on the native ranges . Recentle plants . Correspathogens , were entivities , which mMikania micrantha rhizosphere has a distinct bacteria community structure that is clearly separated from the native plants and the bulk soil. Although some common microbes are observed in the rhizosphere of both M. micrantha and two native plants, M. micrantha rhizosphere specifically recruited Cupriavidus, Enterobacter, Pseudomonas, and Phenylobacterium, which played important roles in resource acquisition, plant hormone regulation, and pathogen suppression. On the other hand, the rhizosphere of native plants P. chinense and P. scandens recruited some other distinctive plant microbes. According to our analysis, the previously found higher soil available P content in M. micrantha rhizosphere was possibly contributed by the enrichment of P-solubilizing bacteria Enterobacter and Pseudomonas. Moreover, pathogens including F. oxysporum and R. solanacearum and pathogenic genes of T3SS were less abundant in M. micrantha rhizosphere compared to the two native plants. In contrast, the biocontrol bacteria such as Catenulispora, Pseudomonas, and Candidatus Entotheonella, as well as the PKS genes were enriched in M. micrantha rhizosphere to develop antibacterial activities. Taken together, these findings deepen our understanding of the microbial composition and function in M. micrantha rhizosphere, as well as the two native plants, and thus provide useful information that would help develop efficient technologies to control the invasion of M. micrantha.SRR8936416–SRR8936475.Publicly available datasets were analyzed in this study. These data can be found in the NCBI under the accession numbers BL, WF, and WQ conceived the study. LY, HeW, YZ, FJ, and SW collected the samples and analyzed the data. YR, CL, HL, WQ, HaW, and FW provided suggestions and helped in the checking. YZ, SW, BL, WQ, and WF helped to revise the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Supplemental Nutrition Assistance Program (SNAP) is the second-largest and most contentious public assistance program administered by the United States government. The media forums where SNAP discourse occurs have changed with the advent of social and web-based media. We used machine learning techniques to characterize media coverage of SNAP over time (1990–2017), between outlets with national readership and those with narrower scopes, and, for a subset of web-based media, by the outlet’s political leaning. We applied structural topic models, a machine learning methodology that categorizes and summarizes large bodies of text that have document-level covariates or metadata, to a corpus of print media retrieved via LexisNexis . For comparison, we complied a separate corpus via web-scrape algorithm of the Google News API (2012–2017), and assigned political alignment metadata to a subset documents according to a recent study of partisanship on social media. A similar procedure was used on a subset of the print media documents that could be matched to the same alignment index. Using linear regression models, we found some, but not all, topics to vary significantly with time, between large and small media outlets, and by political leaning. Our findings offer insights into the polarized and partisan nature of a major social welfare program in the United States, and the possible effects of new media environments on the state of this discourse. The Supplemental Nutrition Assistance Program  is the United States federal government’s primary form of food assistance to lower-income Americans, and is the second-largest welfare program, with a budget of more than $68 billion in 2017 [While the tenor of policymaking debates over SNAP has shifted since the program’s formation in 1964, many key points of dissention are recurrent themes. For instance, frequent debates include whether or not the program disincentivizes work, encourages welfare dependency, or subsidizes unhealthy eating ,4. The tMedia studies have demonstrated how, through the selection and framing of topics, the news media acts as an agenda-setter—impacting the salience of issues and influencing public opinion and policymakers alike –10. SeveTypically, content analyses of print journalism have included a sample of articles published in major newspapers; however, with the advent of online journalism and social media, an increasing number of individuals access news via digital platforms. Digital news sources now play a major role in the media sector; a 2016 Pew Research Center survey about news use found that 38% of Americans reported getting news from digital news platforms, which was second only to those who got their news from TV sources . As the This study used a machine learning technique, structural topic modeling, to assess when, where, and how SNAP has been covered by news media. In particular, we were interested in understanding how SNAP was portrayed by different types of news media outlets and considering how media discourse on SNAP could affect public opinion and policymaking related to this large social welfare program.This study was completed in three general steps: 1) assembling corpora of online and print media about SNAP, 2) refining and preparing these databases for statistical analyses using structural topic models to account for possible temporal variation in topics , and 3) A corpus of print news media  was assembled from a series of queries to the LexisNexis Academic Newspaper Stories/Combined Papers archive, which includes 2,702 major news publications in the United States and elsewhere. Given the size of the archive, we applied simple primary search terms: “food stamp,” “SNAP,” or “Supplemental Nutrition Assistance Program,” and we also included items classified by LexisNexis under the index term “Food Stamps.” We limited the search to articles published between January 1, 1990 and December 31, 2017.tidyr package [snap” or “Stamp out hunger”). To our knowledge, the research procedures described above complied with the terms of service for websites from which data were collected.To create an online media corpus , we used a Google News web-scraping algorithm based on several broad search keywords to include all mentions of SNAP in online media from 1990 to the present. Primary inclusion terms were: “food stamps,” “SNAP,” and “Supplemental Nutrition Assistance Program,” with additional search terms to identify media that uses state-specific program names . A full list of search terms is available in the Supplemental Materials . Additio package ) to remoThe initial search queries within the LexisNexis database generated 79,481 documents. Of these, 58,778 documents were included in the Print Corpus. For the Online Corpus, the initial web-scrape collected 13,987 documents, and ultimately 3,850 documents were included in the final corpus. stm R package [With functions available in the  package , severalTo allow qualitative investigation of change over time, as well as comparison between media sources and outlets, various document metadata were attached to all documents. For both the Print and Online Corpora, the date of publication was included as metadata, as well as the source’s status as a “Major Paper” or “Other Paper” based on the URLs of the 50 most widely-circulated print newspaper URLs, as reported in a recent Pew Research Center report . This ala priori. Alternatively, unsupervised learning analyzes patterns within a text corpus to identify a given number of themes or categories. This approach is ideal for handling large datasets that do not inherently have well-defined categories, and/or would be prohibitively costly in terms of time or other resources to pre-classify. Here, we apply a particular unsupervised machine learning approach, the “structural topic model,” which has been previously used in analyses of large text databases related to political discourse and attitudes [stm, allows for automated selection of optimal model parameters within a set number of topics, and enables flexible estimation of metadata effects  on topic prevalence [Machine learning methods are generally characterized in one of two ways: supervised or unsupervised analysis. In supervised machine learning analyses, pre-classified documents or data are used to “train” a computer algorithm that can then classify new, un-classified data. These analyses can be useful when distinct categories or typologies are known, ttitudes . A specievalence .θ), whereby each document is associated with n estimates, where n is the number of topics in the model. These estimates can be used to assign topics to documents or assess the general distribution of topics across a corpus. The manyTopics function (part of the stm package) identifies an optimal model for a given number of topics, though inspection of average topic semantic coherence  and exclusivity  for each model was also used to assess the optimal number of topics across all models in a corpus.One of the primary outputs of structural topic models are estimates of topic proportions for each document (θ), and documents were assigned to topics by their maximum θ value. The stm package helps summarize each topic by representative words according to several measures: probability, FREX , “lift” , and “score”  [θ values for a given topic were also inspected to better understand the topics indicated by the representative words. The estimated prevalence of topics and effects of the various metadata were measured with linear regressions fit by the estimateEffects function of stm [For both the Print and Online Corpora, a series of structural topic models were generated for three to ten possible topics with the date of publication and a binary variable for major newspapers (based on the aforementioned Pew study ) include topics) . Documenn of stm . All staEight-topic models were selected for the Print, Print Subset and Online Corpora, based on their average topic semantic coherence and exclusivity, compared to models with higher or lower numbers of topics. These values for all numbers of topics are provided as Supplemental Material .The average prevalence of topics across Print Corpus documents ranged from 4%  to 21% . The average year of publication was 2008 (SD = 7.4), and 20% of documents were from newspapers with national readership. Among the Print Subset corpus, the average prevalence ranged from 5%  to 17% , and the average political alignment score was -0.34 (SD = 0.19). Online media topics varied in prevalence from an average of 9% , to 15% . The average year of publication for Online Corpus documents was 2015 (SD = 1.3), 22% of documents were from major news outlets, and the average political alignment score was -0.14 (SD = 0.55).To explore the variance of topic proportions over time, both unadjusted and adjusted regression models were fit with parameters from the structural topic models. The expected prevalence of topics was observed to significantly vary between time intervals in all corpora, adjusting for average political alignment (for the Print Subset and Online corpora only) and major news outlet status (for the Print and Online corpora). All topics were found to vary significantly between time periods in the Print Corpus and Print Subset corpora, and five of eight topics had significant time variation in the Online Corpus. Significant differences between major and non-major media outlets were also observed. Among the Print Corpus, documents from major newspapers had significantly more coverage for half of the topics, and significantly less coverage for the remaining topics. Major media outlets were less predictive of Online Corpus topics and were significantly associated with less coverage for two topics, and more coverage for three topics. For the Online Corpus, the average political alignment of a media source was significantly associated with expected topic coverage: three topics were significantly associated with right-leaning outlets, and five topics with left-leaning outlets. For the Print Subset Corpus, four topics were significantly associated with right-leaning outlets, and one with left-leaning outlets. θ values for Print documents were 0.53 (SD = 0.17), 0.48 (SD = 0.15) for Print Subset documents, and 0.49 (SD = 0.14) for Online documents. For “topic-dedicated” documents with a maximum θ value of 0.50 or greater , a similar distribution is evident between the Print, Print Subset and Online Corpora . Among these more focused documents in the Print Corpus, 23% were classified as Print Topic 8 ; in the Print Subset Corpus, 20% were classified as Print Subset Topic 8 , and in the Online Corpus, 27% were classified as Online Topic 1 . On average, maximum topic Overall, 46% of Print Media documents predominantly pertained to a single topic (“topic-dedicated”), compared to 37% of Print Subset documents and 42% of Online documents. This indicates that the majority of documents included multiple topics, with a possibility of some documents having almost an equal split of two or more topics. Among certain topics, however, the average document’s maximum theta value was much higher; for example, approximately 77% of the documents classified as Online Topic 1  were topic-dedicated, but only 20% of Online Topic 7  documents were similarly topic-dedicated, indicating that the topic more often co-occurred with other topics. These patterns are further detailed in Our findings show how the discourse around SNAP in print and online media significantly varies based on when and where news is reported. Below we highlight two key findings, which may have particularly implications for the broader political discourse and the public perception of SNAP: 1) changes in topic coverage of SNAP over time and in print versus online media, and 2) thematic reporting on SNAP by partisan leaning of news source.In both the print and online media, we found significant variations over time in topic coverage around SNAP see . These fAs other media content studies have indicated, coverage and framing of a policy issue is often different before versus after a major policy shift . Not surGiven that SNAP is an income-eligible entitlement program used by one in seven Americans ,23, it iHowever, shifts in SNAP topic coverage are not just reactions to current events. Given what is known about how media outlets, both print and online, act as agenda-setters for public discourse and for policymakers –10,24, tBased on our analysis, it is evident that the political leaning of media outlets influences the focus of the discussion around SNAP. Within the Online Corpus, all of the topic areas received significantly more coverage in either conservative or liberal media outlets see . SubstanWhile public dislike of welfare has been previously documented among Americans ,12, receCurrently the largest federal food assistance program and one of the largest forms of federally funded financial assistance, SNAP has persistently been the focus of fiscal conservatives. The debate around SNAP has been laden with many of the same contentious issues as with conventional cash welfare including the deservingness of recipients and dependency or abuse of the program by the poor ,26. The One major limitation to this study is its chronological focus (1990–2017). While out of necessity given the nature of archived online content and web-scraping technologies, this does limit our ability to monitor changes in topics over broader time horizons. Nevertheless, both the magnitude and reach of online media sources  underscores and mirrors broader shifts in the media landscape, and reinforces our interest in online-accessible documents. Additionally, structural topic models are but one method of assessing large qualitative datasets, and, given their reliance on machine learning, may not always produce externally valid results. However, because we reviewed and qualitatively assessed model outputs, thus refining and enhancing our media corpora based on preliminary findings, we believe that the final topics described have a higher degree of validity.We attempted to document the various ways in which the Supplemental Nutrition Assistance Program has been represented in news media during the last several decades. Our findings illustrate how the current era of online media complicates previous content analysis strategies. To adapt to these challenges, we employed a topic modeling procedure that was sensitive to changes over time in order to find internally and externally valid topics. In an eight-topic model, themes included a variety of social, political, and economic issues, with expected topic proportions ranging from approximately 9 to 21 percent, and wide variability for certain topics over time. We also found that SNAP coverage fluctuates or remains steady depending on the topic, and while most news media documents featured at least partial representation of multiple topics, some topics, especially fraud, frequently dominated the documents in which they appeared. Combined with the significant political alignment of most topics, we conclude that certain types of SNAP media coverage are both highly partisan in nature and stand relatively apart from the broader media ecosystem.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(CSV)Click here for additional data file.S3 File(CSV)Click here for additional data file.S4 File(CSV)Click here for additional data file.S5 File(R)Click here for additional data file.