Medulloblastoma (MB) is a malignant, heterogenous brain tumor. Advances in molecular profiling have led to identifying four molecular subgroups of MB , each with distinct clinical behaviors. We hypothesize that (1) aggressive MB tumors, growing heterogeneously, induce pronounced local structural deformations in the surrounding parenchyma, and (b) these local deformations as captured on Gadolinium (Gd)-enhanced-T1w MRI are independently associated with molecular subgroups, as well as overall survival in MB patients.1w scan for every patient was registered to a normal age-specific T1w-MRI template via deformable registration. Following patient-atlas registration, local structural deformations in the brain parenchyma were obtained for every patient by computing statistics from deformation magnitudes obtained from every 5mm annular region, 0 < d < 60 mm, where d is the distance from the tumor infiltrating edge.In this work, a total of 88 MB studies from 2 institutions were analyzed. Following tumor delineation, Gd-TMulti-class comparison via ANOVA yielded significant differences between deformation magnitudes obtained for Group 3, Group 4, and SHH molecular subgroups, observed up to 60-mm outside the tumor edge. Additionally, Kaplan-Meier survival analysis showed that the local deformation statistics, combined with the current clinical risk-stratification approaches (molecular subgroup information and Chang’s classification), could identify significant differences between high-risk and low-risk survival groups, achieving better performance results than using any of these approaches individually.These preliminary findings suggest there exists significant association of our tumor-induced deformation descriptor with overall survival in MB, and that there could be an added value in using the proposed radiomic descriptor along with the current risk classification approaches, towards more reliable risk assessment in pediatric MB. CurrentOver the last decade, there has been international consensus  on the g1w) sequences. Recent studies have shown the promise of radiomic (high throughput feature extraction) features extracted from routine MRI scans in capturing the underlying tumor histological and molecular characteristics in MB  including gadolinium-based contrast agent . Hence, the impact of the tumor is observed not just within the visible tumor boundaries, but also in the immediate peri-tumoral, as well as in seemingly normal-appearing adjacent regions . For instance, Group 3 tumors that have relatively poor survival outcomes, are shown to proliferate at a faster rate than WNT subgroup which is associated with improved prognosis –17, sugg1w MRI scans. Our work is based on the rationale that the differential growth patterns across MB molecular subgroups  could be quantified using the local structural deformations in the BAT region and may exhibit distinct deformation patterns across the subgroups. Specifically, we hypothesize that (1) the growth pattern of the more aggressive and heterogeneous MB tumors will exert more pronounced structural deformations on the surrounding BAT regions, as compared to less aggressive MB tumors, and (2) these distinct local deformation attributes from the normal brain parenchyma, as captured on Gd-T1w MRI, will be independently associated with the four molecular subgroups (that vary in the degree of aggressiveness to some extent), as well as overall survival in MB patients. In this work, we present a novel deformation-based radiomic descriptor that captures local structural deformations in the BAT region of pediatric MB patients on routine Gd-T22.11w MRI sequences with (1) acceptable diagnostic quality (as identified by collaborating radiologists), and (2) available overall survival information. Specifically, a total of 10 cases were excluded from the analysis; one case was excluded due to poor image quality during acquisition, one was excluded due to the lack of clinical information, and the other 8 cases were excluded because of the missing protocols that were needed to conduct the analysis . This ultimately resulted in a cohort of n=88 cases (47 from CHLA and 41 from CCHMC), of which n=71 cases had the subgroup information available . Our retrospective study utilized Gd-T1w MRI sequences from pediatric MB patients from two collaborating institutions. The cohort from Children’s Hospital of Los Angeles (CHLA) consisted of 52 studies (mean age = 5.46 years) and the cohort from Cincinnati Children’s Hospital Medical Center (CCHMC) consisted of 46 studies (mean age = 5.28 years). The sequences used in this study were acquired using both 1.5 and 3 Tesla scanners using the gradient recalled (GR) echo sequence. Due to the small and disproportionate number of cases across each of the molecular subgroups from the two institutions , we incorporated the entire cohort of n=71 patients (for whom the molecular information was available) for unsupervised statistical analysis, i.e., without holding out a validation set. Further, in our survival analysis experiment, the disproportionate distribution of MB subtypes was accounted for by dividing the entire cohort of n=88 studies into training and test set using a 70-30 split; following the essential caveat that all MB subgroups are proportionally represented across the training and test sets.2.2via a hand-annotation tool in 3D Slicer. Expert 1 (V.S), a board-certified neuro-radiologist with 10 years of experience, supervised expert 2 , in annotating the MRI slices into the following 2 regions: (1) infiltrating T2/FLAIR hyperintensities (edema), and (2) the normal adjacent region that exists around the infiltrating tumor margins as BAT.For every study used in this work, Gd-T1w MR images were corrected for inhomogeneities caused by the magnetic field using N4 bias correction, followed by applying a histogram matching method for intensity standardization as described in . Specifi2.3To capture structural deformations in the BAT region, all Gd-T1w images of the diseased subject brains were registered to age-appropriate atlases that were obtained from the neurodevelopmental MRI database . SpecifiFinally, to capture localized deformation changes around the infiltrating edge, the region outside the infiltrating T2/FLAIR hyperintensities was divided into equidistant 12 annular bands. These bands were created to be 5mm apart from each other, covering 60 mm outside the tumor margins. The choice for the size of the annular bands was based on previous studies which recommended 5mm as the safe clinical target volume margin for MB tumors . The def2.4Experiment 1: Identifying associations of structural deformation features with MB molecular subgroups using ANOVApost hoc test, ‘multiple comparison of means’, to identify if the means of each of the subgroups have significant statistical differences from other MB subgroups . Highly correlated deformation features from across each of the 12 bands were discarded, and the remaining features were scaled and normalized. A multiclass ANOVA test was used to obtain statistically significant differences in deformation features across the subgroups. This was followed by a ubgroups .Additionally, we conducted a comparative experiment in light of the work by Dasgupta et al. , where wExperiment 2: Identifying the added-value of structural deformation features for MB survival risk-stratification in conjunction with existing approaches, and comparison with clinical variablesFirst, to evaluate the associations of the local deformation features in the context of survival analysis in MB, we employed a least absolute shrinkage and selection operator (LASSO) regression model , along wAdditionally, to assess the performance of RRS and the added value of combining it with the current stratification approaches for pediatric MB, we performed the following analyses, for MB survival prognostication: (1) RRS alone, (2) RRS + Chang’s stratification, (3) RRS + molecular subgroup information, and (4) RRS + Chang’s stratification + molecular subgroup information. For our multivariate cox proportional hazards models, we reported multiple hazard ratios as we obtained distinct hazard ratios for each stratum of every independent covariate. Within each covariate, one stratum will have the ‘baseline’ hazard ratio of 1 against which the other strata are compared. A hazard ratio > 1 indicates increased hazard while a hazard ratio <1 indicates a lower hazard than the baseline strata.Further, for sake of completeness, we utilized the available clinical information for our participating cohorts, to assess their performance in survival prediction on our cohort. Namely, the following parameters were individually employed, i.e., in a univariate setting, into our survival model: (1) Chang’s stratification, (2) molecular subgroup information, (3) tumor volume, (4) EOR, and (5) presence of metastasis.Experiment 3: Assessing associations between RRS and existing risk-stratification approaches via both stratification approaches. Similarly, for Chi-square test, a 4 x 2 contingency table was constructed for comparing predictions from each of the 4 molecular sub-types as well as RRS. For both tests, the chi-squared statistic and the associated p-values were reported.To assess if there exist any statistically significant relationships between the survival predictions of RRS versus the current stratification approaches, we performed the following experiments: (a) McNemar’s test for correlated proportions on RRS outcomes and Chang’s stratification outcomes , and (b) Chi-square test for independence between RRS and molecular subgroup stratification. For McNemar’s test, a 2 x 2 contingency table was constructed based on the predictions of low-, and high-risk groups 33.115), at 20 mm (SK20), and at 25 mm (SK25), between Group 3 vs. SHH and Group 4. Additionally, kurtosis of deformation showed significant differences between the molecular subgroups, where K15 was statistically significantly different between Group 3 and Group 4, and K25 was statistically significantly different between Group 3 vs. SHH and Group 4. Another top feature was median of deformation, where MD25 was statistically significantly different between Group 3 and Group 4. Our analysis pertaining to the association of tumor location-specific semantic features with molecular sub-types yielded statistically significant association between Group 4 tumors and midline horizontal location  as well as between Group 4 tumors and presence of brainstem involvement . Additionally, we identified statistically significant associations between Group 3 tumors and high contrast uptake , and a statistically significant association between SHH tumors and medium contrast uptake .3.2Employing deformation features alone (RRS): When applied on the training set, LASSO survival analysis yielded 3 top deformation features . The KM curve obtained for the training set based on these features  of 5.9. However, for the test set, significant differences were not observed between the low-risk and high-risk survival groups (10) are shown in Qualitative differences for the deformation fields for two MB subjects, one with poor-survival (top row) and one with prolonged survival (bottom row), are shown in Employing clinical features in a univariate setting for survival analysis: When Chang’s classification (n = 88), molecular subgroup information (n = 71), EOR (n =88), presence of metastatic disease (n = 88), and tumor volume (n = 88) were individually employed to survival analysis in a univariate setting, significant differences were not observed between the two survival risk groups. Employing RRS with clinical features in a multivariate setting for survival analysis:(a) RRS + Chang’s stratification: Significant differences were identified between the two risk groups of the training cohort . However, RRS + Chang’s stratification did not yield significant differences between the low-risk and high-risk groups on the test cohort .(b) RRS + molecular subgroup information: Significant differences were observed between the two risk groups of the training cohort using this RRS + molecular subgroup information , demonstrating an improvement in C-index value from the model in (a). While the RRS + molecular subgroup analysis, similarly, did not show significant differences on the test cohort , substantial improvement in the C-index (0.81) was observed on the test set, compared to the values obtained when employing either RRS or molecular information in a univariate setting.(c) RRS + Chang’s stratification + molecular subgroup information: Significant differences were observed between the two risk groups on the training cohort . While the RRS + Chang’s stratification + molecular subgroup analysis did not significant differences on the test cohort, substantial improvement in the C-index (0.792) was observed on the test set, compared to the values obtained when employing either RRS, molecular information, or Chang stratification, in a univariate setting.3.3McNemar’s test for correlated proportions on RRS outcomes and Chang’s stratification outcomes did not yield statistical significance (p = 0.44), suggesting that the predictions of the two models do not hold significant associations between their assessments. Similarly, the chi-squared test between survival predictions of the RRS and molecular sub-group analysis did not yield significant associations (p = 0.86), suggesting that RRS may provide complementary risk-assessment, independent of the existing approaches. The associated contingency tables for the two tests are provided in supplementary documentation.4Medulloblastoma (MB) is an aggressive brain tumor in children, that can have both local mass effect as well as potential for metastasis through the CNS. Locally, this results in variable degree of normal brain parenchymal displacement, that can ultimately lead to worse prognosis and poor survival . In thisFurther, previous studies have employed radiomic and deep-learning approaches to distinguish molecular subgroups of MB using features obtained from within the visible peri-tumor confines alone , 29, 30.The analysis conducted in our work was limited to Gd-T1w images because the well-curated age-appropriate pediatric database  only incApart from identifying MB subgroups, previous works in the context of MB survival analysis have investigated clinical and biological predictors or gene expression profiling analysis , 35. MorIn the context of our presented survival analysis, kurtosis and median of the deformation descriptor were identified as top features by the LASSO regression model, on the training cohort. Interestingly, kurtosis of deformation at 10 mm was statistically significantly different between high- and low-risk survival categories Figure 6Additionally, when combining our deformation descriptor with the current risk assessment approaches that are used in a clinical setting , substantially improved performance metrics  were obtained for survival risk-assessment, compared to any of these three approaches in a univariate setting. This demonstrates the added value of combining the proposed radiomic descriptor with the current stratification approaches, towards more reliable risk assessment in pediatric MB.Our study did have some limitations. Our results were limited to identifying significant associations of deformation features with molecular subgroups and overall survival using a relatively small cohort of retrospective studies (from 2 institutions) and will need to be validated on a larger multi-institutional cohort. Further, when employing the RRS survival model, either alone, or combined with the current clinical stratification approaches (molecular subgroup information and Chang’s classification system), significant differences were not observed between the two risk groups, on the test set, on account of the limited sample size (n = 28 for models involving RRS or RRS + Chang’s and n = 21 for the models that involved molecular information). In fact, it is well documented that larger sample sizes are required for a multivariate regression model to fit well to the data (~10-15 cases per covariate is recommended) , which wOur future work will aim to apply our proposed deformation descriptor to a group of uniformly treated patients with pediatric medulloblastoma. In addition, a larger cohort that evaluates progression free survival  will be employed, to further demonstrate the importance of RRS, complementary to, and in conjunction with existing clinical prognostic markers (EOR. presence of metastasis), as well as Chang and molecular stratification. Also, we aim to integrate deformation descriptors with radiomic texture and shape features from the tumor regions using multi-parametric MRI sequences  for molecular and survival risk-stratification. Further, to ensure clinical utility of our approaches, we aim to build an automated segmentation pipeline for pediatric brain tumors, utilizing our currently curated dataset that includes carefully annotated tumor sub-compartments by expert neuro-radiologists. We also will seek to investigate other clinical attributes as manifested on imaging, such as CSF seeding, and incorporating them as complementary features to our deformation descriptor, for MB survival prognostication and subgroup stratification. Lastly, we will seek to include pathway-specific information in our curated studies, to establish associations between signaling pathways and our deformation descriptor in MB tumors.1w protocol with overall survival in MB patients. Following rigorous prospective multi-site evaluation, the deformation features associated with molecular subtypes could provide complementary information beyond molecular and histological phenotypes towards reliable survival risk stratification and help guide therapeutic treatment management in MB patients.To conclude, in this preliminary feasibility study, we identified associations between our local tissue deformation features extracted from routine Gd-TThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.SI contributed to analysis and interpretation of data, contributed to designing the experiments and the software, helped drafting the original article. MI contributed to the conceptualization, analysis, experimental design and software, and interpretation of data, wrote the original draft and continued revising the article based on co-authors’ feedback. BT provided clinical datasets, led the conceptualization, and helped define the clinical problem and provided clinical interpretation of findings. RS provided clinical datasets, supported the conceptualization, and helped define the clinical problem and provided clinical interpretation of findings. PB and ASM helped curating the clinical datasets and helped with clinical interpretation of findings. RC and JC helped curating the clinical datasets and performed the annotations on radiological images. KB performed the annotations on radiological images. VS supervised all the annotations and corrected them. M-LH supported the conceptualization of the clinical problem. PV and RV contributed to the analysis. DH and AJ helped curating the clinical datasets and helped with clinical interpretation of findings. AM revised the manuscript critically for important intellectual content. PT contributed to the interpretation of the results, contributed to designing the experiments and conceptualization, and supervised the writing and edited the article. PF helped with the design of all the conducted statistical tests. All authors have reviewed the manuscript. All authors read and approved the final manuscript.


The study aimed to prepare ciprofloxacin-loaded polyvinylpyrrolidone electrospun nanofibers for oral drug delivery, using a conventional nozzle-based and a lab-built nozzle-free electrospinning equipment. To produce nanofibers, electrospinning is the process most often used. However, from the industry’s point of view, conventional electrospinning does not have sufficiently high productivity. By omitting the nozzle, productivity can be increased, and so the development of nozzle-free processes is worthwhile. In this study, a solution of ciprofloxacin and polyvinylpyrrolidone was electrospun under similar conditions, using both single-nozzle and nozzle-free methods. The two electrospinning methods were compared by investigating the morphological and physicochemical properties, homogeneity, in vitro drug release, and cytotoxicity. The stability of the nanofibers was monitored from different aspects in a 26 month stability study. The results showed that the use of the nozzle-free electrospinning was preferable due to a higher throughput, improved homogeneity, and the enhanced stability of nanofiber mats, compared to the nozzle-based method. Nevertheless, fast dissolving nanofibers loaded with poorly water-soluble ciprofloxacin were produced by both electrospinning methods. The beneficial properties of these nanofibers can be exploited in innovative drug development; e.g., nanofibers can be formulated into orodispersible films or per os tablets. The pharmaceutical field is facing a tremendous challenge concerning the formulation of poorly water-soluble drugs into effective dosage forms of high bioavailability . To overThe simplest method for drug loading into nanofibers is by dissolving or suspending the drug and polymer in a solvent or mixture of solvents, and electrospinning them together. The conventional single-nozzle ES setup contains a high-voltage power supply, a nozzle, a collector, and a polymer solution or melt filled in a syringe . As the The easiest way to increase the productivity of the ES process is to multiply the number of nozzles, which is called multi-nozzle ES ,16. HoweA model drug, ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic, was used in this study. From a chemical point of view, CIP is a zwitterion that possesses a base function (pKa 6.0) and an acid function (pKa 8.8) with an isoelectric point of 7.4 at 25 °C. As a zwitterion, CIP has a pH-dependent solubility with a minimum near to the isoelectric point. The solubilities of CIP in pH 6.8 and 7.4 phosphate buffer solution (PBS) were reported to be 0.17 and 0.16 mg/mL, respectively . SimilarPolyvinylpyrrolidone (PVP) or povidone is one of the most commonly utilized pharmaceutical polymers. It can be found as a binder in traditional pharmaceutical products such as tablets, but it is also widely used in nanotechnology as a drug delivery polymer. PVP is in favor because it is a non-toxic, biocompatible, biodegradable polymer that is easy to handle . In addiStability is always a key issue for drug delivery nanosystems. There are some stability studies on electrospun nanofibers in the literature, most of which focus only on the crystal structure of the drug . Few of According to the authors’ best knowledge, there is no stability study of electrospun materials regarding the morphology, crystallinity, and the in vitro dissolution of the nanofibers, or a stability study investigating a time period of more than 1 year.In the present study, nozzle-based and nozzle-free ES setups were used to prepare CIP-loaded nanofibers. The effects of the production method on the nanofiber morphology, fiber diameter distribution, structural characteristics, homogeneity, in vitro drug release, and cytotoxicity were studied and compared. Special attention has been paid to the stability of the nanofibers. The 26 month study included not only crystallographic, but also morphological and in vitro drug release experiments.® 250 mg, KRKA, d. d., Novo mesto, Slovenia) were used. Acetonitrile was purchased from Molar Chemicals . The thiazolyl blue tetrazolium bromide (MTT) reagent and sodium dodecyl sulfate were obtained from Sigma-Aldrich . Phosphate buffer solutions  were prepared in-house. All other chemicals were analytical grade, and purified water was used.Ciprofloxacin powder  was donated by Teva Pharmaceutical Works Ltd.  for research work. Polyvinylpyrrolidone  was purchased from Alfa Aesar . The solvents for the ES solutions, the ethanol (99.99% purity), and the glacial acetic acid were obtained from Fisher Scientific  and Sigma-Aldrich , respectively. As a reference for the in vitro dissolution studies, commercially available CIP-containing filmcoated per os tablets  with a cone-plate configuration. The diameter of the cone was 60 mm, and the angle was 1°. The temperature of the cone and plate were kept at 21.0 °C. The apparent viscosity of the ES solutions was calculated as the average value ± S.D. under the shear rate range of 31.85–100 s−1.For the electrospinning solutions, firstly, the drug and polymer solutions were prepared separately. Polymer solutions of 5 and 10 A commercially available, IME electrospinning device  was used as a single-nozzle setup for nanofiber production A. In ourThe nozzle-based ES setup was equipped with a stainless-steel 20-gauge nozzle, as the tip of a 2 mL syringe filled with the electrospinning solution and was connected to a syringe pump . The nozzle was clamped to the positive electrode of a high-voltage power supply generating +20 kV. An aluminum disk was used as a collector, and it was charged with −4 kV. The nozzle-collector distance was set at 15 cm and the flow rate was kept at 1 mL/min. The chamber of the setup was at room temperature, with a relative humidity of 31–36%.The nozzle-free ES apparatus was built in-house B. The deThe names and the differences of the nanofibrous samples prepared both with nozzle-based and nozzle-free ES equipment are listed in w/w% and 10 w/w% CIP powder. The physical mixtures were homogenized using a shaker mixer  under controlled conditions .Physical mixtures were used for the DSC and XRPD measurements as reference samples. The mixtures contained PVP powder, and 5 The morphological appearance of the electrospun fibers was investigated via scanning electron microscopy  at 10 kV. The samples were previously coated with a thin (approximately 10 nm) film of gold–palladium using a sputter coater . The SEM images were used to measure the diameters of the nanofibers. A total of 50 nanofibers from each formulation were selected, and the mean fiber diameter was measured using ImageJ 1.44p software .Differential scanning calorimetry  was applied to evaluate the thermal behavior of the samples. Approximately 3–5 mg of the nanofibrous samples were measured in the temperature interval of 30–300 °C at a heating rate of 5 °C/min, under a constant argon flow of 150 mL/min.−1 and with a resolution of 4 cm−1.Fourier transform infrared spectroscopy  was performed after discs of a KBr and nanofibers had been made. The samples were ground with 150 mg dry KBr in a mortar and pestle, then compressed into a disc with 10 t pressure. The discs were scanned 128 times over the range of 4000–400 cmThe crystalline s of the nanofibers, the physical mixtures, and the raw drug were characterized using an X-ray powder diffraction system . The samples were measured with Cu Kα radiation (λ = 1.5406 Å), scanned at 40 kV and 40 mA in the interval of 3–40 2θ, and a VÅNTEC-1 detector was used. All manipulations  were performed with DIFFRAC plus EVA software .2) were cut from the edge and the center of the collector. Raman spectra of the solid specimens were recorded with a Thermo Fisher DXR dispersive Raman microscope  equipped with a CCD camera and a diode laser operating at a wavelength of 780 nm. The applied laser power was 24 mW at a slit aperture size of 50 µm. The spectra were collected from a 400 × 400 µm2 area with a 50 µm step size in the spectral range of 200–3300 cm−1. The exposure time was 6 s and an average of 8 scans was applied to construct Raman chemical map. Determining the distribution of CIP in the specimens, the spectral range from 1650 to 1550 cm−1 was selected for profiling  and entrapment efficiency (EE) of the specimens collected from the edge and the center of the collector were also investigated. The DL and EE were quantified spectrophotometrically  at 271 nm after dissolving the specimens in pH 7.4 PBS.DL and EE of the nanofibrous formulations were calculated according to the following equations:2 specimens. After the dissolution of the specimens in pH 7.4 PBS, the measured mass of CIP was calculated from the UV absorption of the drug.The nanofiber weight was obtained by weighing the 0.5 × 0.5 cmThe measured CIP concentration was calculated from the UV absorption of CIP after weighing and dissolving the specimens in pH 7.4 PBS. In addition, the initial CIP concentration in the electrospinning solution was considered as the total CIP content.Each experiment was performed in three parallel measurements, and the average values and standard deviations are reported.® 250 mg tablets. The release studies were carried out in 50 mL of pH 7.4 PBS medium at 37 °C. The paddle was rotated at 100 rpm. Samples of 0.5 mL volume were taken manually from the buffer solution after 1, 3, 5, 10, 15, 20, 30, 40, 50, 60, and 90 min. After sampling, the volume was replaced with fresh PBS.As part of the comparison of the nozzle-based and the nozzle-free ES, in vitro drug-release studies were carried out. A modified paddle method  was used to measure the drug release from the different nanofibers (containing 25 mg CIP) compared with 25 mg raw CIP powder, and Ciprinol® C18  analytical column at 25 °C. The mobile phase consisted of (A) an aqueous solution of 0.02 M KH2PO4 adjusted to pH = 2.8 with ortho-phosphoric acid and (B) acetonitrile with isocratic elution  at a flow rate of 1 mL/min. The analytical time was 5 min for each injection. A sample volume of 20 μL was injected to determine the concentration of CIP at 210 nm, using a UV/VIS diode array detector. Data were evaluated using ChemStation B.04.03. Software . The retention time of the drug was 3.57 min (2) of the calibration curve was 0.998.The concentration of the drug present in the aliquots was determined with high performance liquid chromatography . Chromatographic separation was performed using a Kromasil3.57 min . The con3.57 min . The reg2 was accepted as the release kinetics.The release kinetics of CIP from the nanofibers were determined. The fitting of the results was tested with zero order, first order, Higuchi, the Korsmeyer–Peppas model, and the Hixson–Crowell cube root law . The modThe nanofibrous samples were stored in a desiccator, protected from light, at room temperature for 26 months. Stability was tested after 3, 5, 8, 16, and 26 months of storage. In order to obtain external and internal information regarding the CIP-loaded nanofibers, their macrostructure was investigated using SEM images and ImageJ software, and their microstructure via XRPD. Then, in vitro drug-release studies were performed on the samples considered critical, i.e., at 8 and 16 months. The methods for the measurements were described above.4 cells/well. Firstly, serial dilution was made of the following stock concentrations: 325 µg/mL ciprofloxacin solution, 1570 µg/mL SN-5 solution, 1570 µg/mL SN-10 solution, or 1520 µg/mL NF-5 solution, and then the cells were incubated with the mentioned materials at 37 °C for 24 h. Later, 20 μL of MTT reagent was added to each well. After an additional incubation at 37 °C for 4 h, 100 µL sodium dodecyl sulfate solution (10% in 0.01 M HCI) was added, and the cells were incubated overnight. The cytotoxicity of the compounds was then determined with an EZ READ 400 ELISA reader  by measuring the OD at 550 nm (ref. 630 nm). The assay was repeated four times for each concentration.Mitochondrial activity quantification as a measure of cell viability was performed via MTT assay in 96-well cell culture microplates using Caco-2  cells. Caco-2 cells were seeded at a density of 4 × 10® 250 mg tablet were compared one by one. The results with p < 0.01 were assumed to be statistically significant.Statistical analysis was carried out to determine the existence of a significant difference between the measured data. A one-way analysis of variance (ANOVA) with post hoc Tukey HSD test was performed on the fiber diameter and the in vitro drug-release data. For the latter, all measured points of the nanofibers, the untreated CIP powder, and the CiprinolThe morphology and fiber diameter distribution of the nanofibrous mats were important for the comparison of the conventional nozzle-based ES apparatus and the lab-built nozzle-free ES equipment. During single-nozzle ES, continuous fibers with smooth surfaces were formed, as shown in p < 0.01) compared with SN-5 . The size distribution of the nanofibers was homogeneous, as the standard deviations were small, and the distribution diagrams were monodisperse and bell shaped  appeared on the spectra, as well as the broad peaks of PVP (3700–2800 cm−1). The shifts and widenings confirmed the successful incorporation of the CIP into the nanofibers in all cases, regardless of the ES method.The main interactions between the PVP and the CIP were observed via FTIR spectroscopy. The homogeneity of the nanofiber mats deposited on the collector during the ES was investigated using two different methods. Both methods measured the CIP content in the specimens taken from the center and the edge of the mats.Firstly, Raman chemical mapping was executed on the specimens . The war2 = 0.9990)  and entrapment efficiency (EE) were calculated from the absorbance values, using a calibration curve (R 0.9990) . The resIn summary, nozzle-free production results in a more homogeneous nanofiber mat gathering on the collector. The reason for this may be that the point of origin of the jets is not concentrated at a single point, as in nozzle-based ES, but ejected from several locations of the bath simultaneously.® 250 mg tablet in pH 7.4 PBS. The dissolution curves are shown in w/w% CIP-loaded nanofibers [In vitro releases of CIP from the different nanofibers were compared with the dissolution of untreated drug powder and Ciprinolnofibers .p < 0.01) than the values of the CIP powder or the tablet at almost every sampling point. In the first few minutes of release, it was variable as to whether the difference was significant, but after 5 min, the significance was obvious. Therefore, it can be concluded that a fast and complete release of CIP from all nanofibers was achieved, and that the dissolved percentages of the CIP was significantly higher (p < 0.01) than in the case of the CIP powder or tablet.A statistical analysis of the percentages measured at each time point was performed, to allow a more accurate comparison of the release . There ww/w% CIP (samples SN-5 and NF-5) could be best described using the Korsmeyer–Peppas model and first-order kinetics. This strong fit to the Korsmeyer–Peppas model was also true for the drug release from the 1 w/w% CIP-loaded nanofibers studied previously [2 = 0.9876). The release curves for raw CIP powder and commercially available tablets were best fit using the Higuchi model, although with smaller R2 values of 0.9148 and 0.8243, respectively. Similar to these results, it was observed in our previous experiments that the R2 for the raw CIP was approximately 0.9 for the Higuchi and Korsmeyer–Peppas models, while the same models were fit with a higher R2 value for the nanofibrous formulations [2 values were adequately high.The kinetics of the in vitro drug release were determined. The regression coefficient of the models fit into the release curves can be seen in eviously . Additioulations . The resAs the first step of the stability study, electron microscopy images of the nanofibrous samples stored in the desiccator were taken. As the second step of the stability study, the crystallinity of the CIP was monitored via XRPD measurements. As reference, CIP was dissolved in ethanol and acetic acid, and after crystallization, the powders were used. The characteristic peak of CIP-acetate at around 6 2-Theta was detected in several SN-10 nanofibrous samples, indicating that the drug had partially re-crystallized . At 5 moThe morphology of the fibers can affect the processability and the dissolution of the API. The latter is possibly affected by the crystal structure of the API, as well. For this reason, in vitro drug release was investigated as part of the stability test. In the literature, there are examples for both maintained and changed drug release from nanofibers after 3 months of storage ,55,60. IOverall, in terms of drug release, the nanofibers prepared with nozzle-free ES were found to be more stable, but the limiting factor that the samples were stored in a desiccator should be taken into consideration.The in vitro cytotoxicity was tested using an MTT assay . CaCo-2 Nanofibers as amorphous solid dispersions can be formulated as orodispersed films or oral medicines, while with the suitable polymer, fast disintegration and rapid release can be achieved. This paper presented the production and investigation of CIP-loaded PVP nanofibers prepared via the nozzle-based and nozzle-free ES methods. Nanofibers with 5% and 10% CIP concentrations were fabricated using conventional single-nozzle ES. Using the nozzle-free method, 5% CIP-loaded nanofibrous samples were produced from the same electrospinning solutions. Comparing the nanofibers, we found that the preparation method had no influence on the drug carriers as amorphous solid dispersions, because both methods could amorphize the CIP. Additionally, the presence or absence of the nozzle had no effect on the in vitro drug release, as the dissolution of the CIP from the nanofibers was complete and fast, and occurred according to the Korsmeyer–Peppas model or first-order kinetics. This result was similar to the preliminary study in which CIP was present in lower concentrations in the PVP nanofibers . It alsoHowever, it can be concluded that nozzle-free ES has several advantages. These are, based on the studies performed, a more homogeneous distribution of the active ingredient within the nanofiber mat, a higher encapsulation efficiency, and a longer stability. An additional advantage may be increased productivity, due to the fact that ES from the free solution surface allows the formation of several Taylor cones at the same time. On the other hand, this also leads to the disadvantage of the nozzle-free method, which is a wide variability in fiber diameters.The study also focused on the stability of the nanofibers. In this context, SEM, XRPD, and in vitro release studies were carried out, which led to two types of conclusions. The first conclusion was that aged morphology, the fusion of the fibers or partial re-crystallization of the CIP did not affect the in vitro drug release. Based on these results, it is important to check for drug release in all stability studies of the nanofibers. As a second conclusion, the stability results suggested that NF-5 was sufficiently stable for 16 months in terms of morphology, and for 26 months in terms of amorphousness and in vitro release. In contrast, the morphology of the SN-5 sample remained unchanged up to 26 months, but the CIP started to re-crystallize at 8 months, and its release slowed down significantly at 16 months. Thus, the nanofibers prepared with nozzle-free ES were found to be more stable during the 26 months of storage in a desiccator.Overall, nanofiber production using nozzle-free ES equipment can not only provide a good alternative to the nozzle-based ES, but it also produces nanofibers with improved properties. Thus, it could be worthwhile to develop the method further and to use the resulting nanofibers as drug delivery systems.


Neuroplasticity in Rehabilitation, a Research Topic exploring how the nervous system changes in response to rehabilitative interventions. While the field of human neural interfacing began with the development of bypass devices , botulinum neurotoxin injection , and rapid-reciprocal leg training , to newer techniques like robotic-assisted therapies , biofeedback , and exoskeleton-based walking with trans-spinal stimulation . Similarly, resultant neuroplasticity is measured with a range of classical functional outcome measures  and reflex arc assessments  to newer imaging-based surrogates of brain activation and connectivity such as functional magnetic resonance imaging (fMRI) , diffusion tensor imaging (DTI) , and functional near-infrared spectroscopy (fNIRS). Finally, Proulx et al. round out our topic by providing a thoughtful narrative review on the importance of multimodal sensory feedback in neurorehabilitation strategies, making a case for the importance of intentionally incorporating brain multisensory integration areas during motor learning to improve motor outcomes in stroke.The sheer number of modalities available for rehabilitation and the study of its effects on the human nervous system has rapidly expanded, and this Research Topic represents a sampling of active areas of investigation. Interventions presented here span classical rehabilitation therapies like stepping  repair; instead, it is our persistently incomplete understanding of human CNS organization, regeneration, and plasticity. Because the CNS is such a complex system that functions on physical scales ranging from microscopic/molecular to full human body circuitry and interacts on timescales ranging from near-instantaneous to a lifetime , obtainiHowever, even short of a complete understanding, incremental progress and discoveries are having tangible impacts. For example, we are now seeing unprecedented recovery from spinal cord injury –8, novelAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Recently, an increasing number of studies relied on the assumption that visually induced changes in choroidal thickness can serve as a proxy to predict future axial eye growth. The retinal signals controlling choroidal thickness are, however, not well defined. We have studied the potential roles of dopamine, released from the retina, in the choroidal response in the chicken.Changes in retinal dopamine release and choroidal thickness changes were induced by intravitreal injections of either atropine (250 µg or 360 nMol), atropine combined with a dopamine antagonist, spiperone (500 µMol), or spiperone alone and were tracked by optical coherence tomography (OCT). To visually stimulate dopamine release, other chicks were exposed to flicker light of 1, 10, or 400 Hz (duty cycle 0.2) and choroidal thickness was tracked. In all experiments, dopamine and 3,4-Dihydroxyphenylacetic acid (DOPAC) were measured in vitreous, retina, and choroid by high-performance liquid chromatography with electrochemical detection (HLPC-ED). The distribution of the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and alpha2A adrenoreceptors  was studied in the choroid by immunofluorescence.The choroid thickened strongly in atropine-injected eyes, less so in atropine + spiperone–injected eyes and became thinner over the day in spiperone alone-, vehicle-, or non-injected eyes. Flickering light at 20 lx, both 1 and 10 Hz, prevented diurnal choroidal thinning, compared to 400 Hz, and stimulated retinal dopamine release. Correlation analysis showed that the higher retinal dopamine levels or release, the thicker became the choroid. TH-, nNOS-, VEGF-, and alpha2A adrenoreceptor–positive nerve fibers were localized in the choroid around lacunae and in the walls of blood vessels with colocalization of TH and nNOS, and TH and VEGF.Retinal DOPAC and dopamine levels were positively correlated with choroidal thickness. TH-positive nerve fibers in the choroid were closely associated with peptides known to play a role in myopia development. Findings are in line with the hypothesis that dopamine is related to retinal signals controlling choroidal thickness. To evaluate the potency of interventions to reduce the progession of myopia, axial eye growth needs to be compared in untreated and treated animal models, or children . UnfortuSince the relationship between retinal dopamine release and choroidal thickness appears complex and does not always follow a simple prediction (more dopamine – > choroid thicker), we have performed three further experiments: (1) We tested whether a dopamine antagonist, spiperone, can reduce choroidal thickening, induced by atropine. (2) We tested whether changes in dopamine release, induced by flickering light or by brighter office light, are related to changes in choroidal thickness. In both experiments, we measured the corresponding dopamine and DOPAC levels in the vitreous and retina and, in particular, in the choroid. (3) We performed an immunohistochemical analysis of the distribution of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, in the choroid and studied colocalizations with peptides known to be involved in the visual control of eye growth.One-day-old male White Leghorn chickens were obtained from a local hatchery . They were raised under an 11/13 h light/dark cycle, with the light phase starting at 8:00 a.m. Water and food were supplied ad libitum. Illumination was provided by light bulbs that produced an ambient illuminance of about 500 lx on the cage floor . Animals were studied between post-hatch ages P14 to P21. In total, 70 chickens were studied. All experiments were conducted in accordance with the ARVO statement for the use of animals in ophthalmic and vision research and were approved by the University Committee of Tuebingen for experiments involving animals (AK 01/19G).Intravitreal injections were performed using a 0.5-mL insulin syringe . They consisted of atropine sulfate monohydrate  in 25 µL saline, or spiperone  in 25 µL ascorbic acid , or a combination of both atropine and spiperone in 25 µL ascorbic acid, while the fellow eye received 25 µL saline or 25 µL ascorbic acid, respectively. Alternatively, both eyes received 25 µL saline. The atropine dose was selected based on previous experiments , where dhttps://imagej.nih.gov/ij/). An interobserver correlation analysis for the OCT measurements of choroidal thickness was performed in a previous study in which both observers were blinded with regard to the treatment of the chickens [We had found earlier –30 that chickens  and show2 = 0.98) and good reproducibility in retention time (0.03%); the limit of detection on column was smaller than 1 pg. In retina and choroid, biogenic amine content was determined as nanogram per milligram protein (ng/mg protein), whereas in the vitreous, DOPAC and dopamine amounts were determined relative to wet weight (ng/0.1 g wet weight).Because dopamine levels cycle diurnally , preparaChickens were placed into a cage that was covered with an array of 5 equally spaced strips with 6 white LEDs each  on top. The cage was set up in a dark room so that, except for the flickering light, no light from external sources could enter. Flickering light was generated by a function generator , combined with a 20 W audio amplifier. The function generator was set to provide a square wave signal with 20% duty cycle and 97.7% modulation at either 1, 10, or 400 Hz, generating an average illuminance in the cage of about 20 lx. Frequencies were selected based on findings that low frequencies induce myopia in a variety of animal models . Data are shown as the mean ± standard deviation. A mixed model repeated measure ANOVA (with time as the repeated measures and group as between subject factor) was used for comparisons of mean choroidal thickness among different treatment groups, followed by a Tukey–Kramer honestly significant difference (HSD) test for post hoc analysis. Two-tailed unpaired Student’s We had previously found that intravitreal atropine causes prominent, but transient, choroidal thickening in the chicken eye. The effect was also observed in the contralateral eye where it was more transient . Time cop < 0.05 and p < 0.01, respectively. Nevertheless, the choroid remained still thicker than in ascorbic acid–injected or saline-injected eyes , 217.18 ± 32.48 µm (16:00), and 205.97 ± 13.44 µm (18:00), versus ascorbic acid–injected eye 212.44 ± 27.5 µm (14:00), 183.88 ± 29.07 µm (16:00), and 170.93 ± 29.39 µm (18:00); p < 0.05, p < 0.05, and p < 0.01, respectively). Furthermore, the effects of atropine were clearly visible also in the fellow eyes, as described before [p < 0.01, p < 0.01, and p < 0.05, respectively).Spiperone reduced atropine-induced choroidal thickening Fig. : spiperod before  but the p < 0.01).In control eyes Fig.  but therp < 0.01), compared to the untreated control. The effect must, however, be unspecific since similar changes were observed with ascorbic acid (2.83 ± 1.33 ng/mg protein). In vitreous, combined application of atropine and spiperone reduced DOPAC content (4.64 ± 0.68 ng/0.1gw.w.), compared to atropine alone . Again, a similar effect was observed for ascorbic acid (4.61 ± 1.01 ng/0.1 gw.w.) versus atropine .Atropine had no effect on DOPAC in the retina (1.77 ± 0.9 ng/mg protein) or vitreous .To test further how dopamine release from the retina might be involved in the control of choroidal thickness, we used another way to stimulate dopamine. It is known since 1971  that dopght Fig. . Howeverp < 0.05 and p < 0.01, respectively) even though the illuminance was lower by a factor of 17.5 (green bar). This confirms that flickering light stimulates retinal dopamine more than just elevating illuminances. The pattern was similar in the choroid . Dopamine levels in the retina changed in the same direction as the levels of DOPAC in vitreous and retina  and laboratory light at 470 Hz . It can be summarized that 1 and 10 Hz flickering light exposure for 2 h increased dopamine in retina and choroid, compared to 400 Hz.HPLC measurements confirmed that flickering light stimulated dopamine production in the retina. Both 1 and 10 Hz flickering light changed dopamine and DOPAC in a similar way in all 3 measured tissue layers, vitreous, retina, and choroid  for dopamine although a trend was seen also for DOPAC. Results suggest that the initial choroidal thickening rate was associated with an increase in vitreal DOPAC and dopamine which indicates more dopamine release from the retina [To further evaluate potential links between dopaminergic transmission and choroidal thickness, changes in choroidal thickness at 3 time points  were correlated with the changes in DOPAC and dopamine as measured around noon. It is likely that a correlation that persists across different experiments (atropin/spiperone treatment and flicker experiments) makes a stronger case that both variables are correlated. Correlation analyses included data from both spiperone and flicker experiments. Choroidal thickness data were collected in the same chickens a day before the HPLC data where it was necessary to sacrifice the animals. On both days, chickens had undergone the same treatments . There are a number of partially highly significant correlations among DOPAC and dopamine in the three studied tissues . These correlations are unlikely to be random but rather reflect interactions of DOPAC and dopamine in these layers. Significant correlations were found between choroidal DOPAC and vitreal dopamine , choroidal DOPAC and retinal dopamine , vitreal and retinal dopamine , and vitreal and choroidal dopamine  .Different from observations by Megaw et al. , vitrealDopamine in the retina was correlated with dopamine in the vitreous, dopamine in the choroid, and DOPAC in the choroid Fig. . While tIn the chicken, we localized TH immunoreactivity in varicose nerve fibers and axon bundles which spread over the entire choroid, in the suprachoroid, in choroidal stroma tissue, around lacunae, and in the walls of blood vessels; an overview is shown in Fig. In an earlier study, we had found in chickens that bevacizumab, an anti-VEGF antibody, suppressed choroidal thickening that normally occurs when eyes recover from induced myopia . We locaUsing choroidal thickness changes as a measure of potential retinal output signals that control axial eye growth, we found that (1) spiperone, a D2 antagonist, reduced atropine-induced choroidal thickening and also made the choroid thinner without atropine; (2) choroidal thickness remained thicker under continuous bright light than under flickering light at 1 and 10 Hz and became thinnest at 400 Hz (which was far above the flicker fusion frequency); (3) flickering light at 1 and 10 Hz at 20 lx caused higher retinal and vitreal dopamine levels than continuous light at 470 lx even though it was 15 times brighter and also compared to flickering light at 400 Hz; and (4) dopamine and its metabolite DOPAC were also detected in the choroid . Both vInitially, choroidal thickening was assumed to be independent of dopamine because the choroid still thickened with positive lenses after reserpine or 6-OHDA injections  which seThomson et al.  recently2 = 0.2135, p = 0.02). Choroidal DOPAC to dopamine ratios were not correlated with choroidal thickness. Interestingly, the DOPAC/DA ratio was much higher in the choroid (13.17 ± 7.02), compared to retina .Tian et al.  had statIt has been found before that flickering light stimulates dopamine release from the chicken retina i.e., , 53, 54), 5453, 5p = 0.02), suggesting that changes of dopamine in the choroid may have a function. However, as supported by finding 6 below, it is unlikely that retinal dopamine can diffuse through the RPE [Our HPLC measurements in choroidal tissue clearly showed that both dopamine and DOPAC were present in the choroid. Furthermore, both vitreal and choroidal dopamine concentrations were correlated with retinal dopamine concentrations ( the RPE . Brown e the RPE  state thWhile retinal and choroidal dopamine levels were correlated if data from all experiments of the current study are pooled, only vitreal and retinal dopamine levels are linked by diffusion of dopamine . As descSchroedl et al.  had idenIn an earlier study, we had found in chickens that bevacizumab, an anti-VEGF antibody, suppressed choroidal thickening that normally occurs when eyes recover from induced myopia  and locaFinding 6 suggests that dopamine is also produced in the choroid but its role has not been described. There are a number of agents controlling blood flow and choroidal thickness . StudiesIn the current study, we have localized several of the agents that were previously proposed to be involved in the control of choroidal blood flow and choroidal thickness. They were colocalized with TH, the rate-limiting enzyme of dopamine. We have found close contacts of TH-positive nerve fibers with nNOS- and VEGF-positive nerve fibers, as well as contacts between TH-positive fibers with alpha2A adrenoreceptors in the walls of the blood vessels.Our study is in line with the hypothesis that atropine exerts an effect on choroidal thickness via dopaminergic transmission. We also provide evidence that a dopamine signal is acting in the choroid although previous studies have shown that it does not reach the choroid by diffusion from retinal sources but rather after relay at the RPE through other messengers at the choroidal side of the RPE. Our results expand the knowledge on signals by which the retina controls the thickness of the choroid and are important for the understanding of myopia development.


Kaposi sarcoma is a vascular endothelial neoplasm caused by human herpesvirus 8. Although it is a well-studied disease, little is known about the specific characteristics or epidemiology of Kaposi sarcoma in Afghanistan. The data consist primarily of anecdotal reports and epidemiological studies extrapolated from neighboring countries. In this case series, we summarize existing data about Kaposi sarcoma in Afghanistan and present seven histologically confirmed cases with associated clinical features to shed light on the characteristics of Kaposi sarcoma in this unique geographic setting. Kaposi sarcoma (KS) was first described by Dr. Moritz Kaposi in 1872 as a multifocal, pigmented sarcoma of the skin, representing an uncommon tumor affecting elderly Mediterranean men. Since that time, KS has garnered attention in association with human immunodeficiency virus (HIV) as an acquired immunodeficiency syndrome (AIDS)-defining neoplasm of endothelial cells, caused by human herpesvirus 8 (HHV-8) . OptimalHistologically, KS is characterized by slit-like, vascular spaces which dissect through dermal collagen bundles. The low magnification view of KS is that of a cellular dermal nodule with possible varying degrees of overlying epidermal changes. The dermal proliferation is composed of a spindle-cell proliferation of endothelial cells forming sinuous vascular spaces. These endothelial cells are typically monomorphic with minimal pleomorphism. The presence of the promontory sign may be helpful; however, this finding is not specific to KS. Extravasated red blood cells (RBCs) and hemosiderin deposition are frequent findings ,2. The dHHV-8 immunostaining is performed on formalin-fixed, paraffin-embedded tissue using monoclonal antibodies to viral antigens . The patKS is a well-studied disease, and much data have been reported regarding presentations in many parts of the world ,2,3,4,5.The cases presented here were collected as part of a collaborative teledermatopathology effort between the Maiwand Teaching Hospital Dermatology Department in Kabul, Afghanistan; CURE International Hospital in Kabul, Afghanistan; Emory University in Atlanta, Georgia; and the University of Arkansas for Medical Sciences in Little Rock, Arkansas. In this model, patients were referred to Maiwand for further workup and treatment. Skin biopsies were then sent to CURE International Hospital in Kabul for pathologic diagnosis. The results were documented, photographed, and forwarded to a dermatopathologist at Emory University, who stained the blocks with HHV-8 and provided interpretation and diagnosis. Additional feedback was provided by a dermatopathologist and soft tissue pathologist at the University of Arkansas for Medical Sciences. This type of collaborative effort has been reported previously with beneficial outcomes .Seven patients were evaluated in the Dermatology Department at Maiwand Teaching Hospital, located in Kabul, Afghanistan. The majority (4/7) were from the remote Northern provinces of Afghanistan, and all presented with violaceous nodules and papules in varying distributions. Only one patient tested HIV-positive. Patient demographics, presentation, and histology are summarized in The patient (#1)  is a 63-A 4 mm punch biopsy was obtained from a well-developed nodule of the left leg and showed infiltration of the dermis by slit-like vascular spaces lined by spindled endothelial cells. There was no extravasation of RBCs or significant atypia of the endothelium b,c. A diThe patient (#2)  is a 60-A 4 mm punch biopsy was obtained from a nodule of the right leg and showed fascicles of spindle cells with slit-like spaces. Erythrocytes were present in vessels, but without much extravasation. There was an element of background stasis-change in the superficial dermis b,c. The The patient (#3)  is a 45-A 4 mm punch biopsy of the left lower extremity was obtained and showed dermal nodules of uniform, spindled cells with slit-like vascular spaces. Some ectatic vascular channels are identified, along with eccrine coil involvement. Few extravasated RBCs are identified b,c. A diThe patient (#4)  is a 61-A 4 mm punch biopsy of a representative papule of the left lower leg was obtained and showed orthokeratosis with a dermal nodule composed of vascular channels with a flattened endothelial lining. Endothelial nuclei were bland, and some RBC extravasation was noted b,c. A diThe patient (#5)  is a 68-A 4 mm punch biopsy of the right ankle was obtained and showed a dermal proliferation of small and large blood vessels with some extravasation of erythrocytes. There were some spindle cells that were not overtly atypical b,c. KS wThe patient (#6)  is a 27-A 5 mm punch biopsy from the center of an indurated nodule on the left shoulder showed a proliferation of pleomorphic, plump, spindled cells in the mid-dermis. This lesion demonstrated infiltration of surrounding collagen by slit-like spaces and abundant extravasated RBCs b,c. A diThe patient (#7)  is an 85A 4 mm skin punch biopsy of a nodule on the right lower leg was obtained and revealed closely packed blood vessels lined by spindled endothelial linings with some extravasation of erythrocytes within the dermis b,c. KS wWe found a predominance of KS in men, primarily from the Northern provinces of Afghanistan. The patients presented with characteristic clinical features. Lesions tended to be multifocal , with a predilection for the lower extremities , upper extremities , and oral mucosa . KS lesions were also identified involving the trunk . The majority of patients were older men; one middle-aged woman; and one younger, adult male . Only one of the seven patients tested positive for HIV. This individual’s HIV status was previously unknown, and the clinical suspicion of KS prompted HIV screening.Since the majority of these cases (6/7) are present in AIDS-negative individuals and lack clinical features identified of other subtypes of KS, they are best classified as examples of classic KS. This finding is not unexpected, as classic KS, although rare, is most common in individuals of Mediterranean or Middle Eastern descent . A possiMany of the traditional histologic features of KS were represented. All cases demonstrated classic, endothelial-lined, slit-like spaces. Five of the seven cases exhibited some degree of RBC extravasation—three of which were qualified with descriptors such as “few” or “some”. Two cases did not display any appreciable extravasated RBCs. Features such as eccrine coil involvement, a well-developed promontory sign, and a fascicular endothelial growth pattern were all variably represented. In all cases, a diagnosis of KS was confirmed by demonstrating HHV-8 expression by immunohistochemistry. The patient characteristics are summarized in Based on data from the International Agency for Research on Cancer (IARC), KS has an incidence rate of 0.01/100,000 people per year in Afghanistan. However, this number is derived from averages in surrounding countries, such as Iran and India ,9,10,12.A diagnostic challenge for this population is that general healthcare services and pathology services are severely limited or non-existent in many provinces in Afghanistan. Individuals tend to present in more advanced stages due to a lack of, or inability to seek out, healthcare. Pathology services are centralized and found in more-urban locations; patients from rural provinces are often unable to obtain a histologic diagnosis until they travel to the capital city of Kabul. Travel to any city, let alone Kabul, may prove an insurmountable task for some individuals due to the remoteness of their provinces. This ultimately results in delayed diagnosis and inappropriate treatments.Regarding the diagnosis of KS specifically, one of the barriers in pathology is the lack of availability of diagnostic, immunohistochemical stains, such as that for HHV-8. Immunohistochemical stains generally require a higher volume of cases and appropriate quality controls. Therefore, clinical suspicion and hematoxylin- and eosin-based light microscopy are the most valuable tools. Follow-up for these patients brings its own unique challenges, and waiting for the results of HHV-8 stains may be impractical ,12,13.Telemedicine and telepathology services are burgeoning healthcare-delivery platforms. These services provide opportunities to expand access to healthcare to underserved populations in remote areas, such as developing countries . Access Finally, one patient (#6) was HIV positive with a history of heroin abuse. Little research has been done on illicit drug use in Afghanistan, even though most of the world’s opium originates in the country . OngoingOur interest in this topic was driven by the needs of local healthcare providers and the paucity of published data regarding dermatologic diseases in a largely underserved population. To date, data consist mainly of anecdotal reports and epidemiological studies extrapolated from neighboring countries. This case series represents an international collaboration between dermatologists, general pathologists, dermatopathologists, and soft tissue pathologists across multiple countries. The unique opportunities afforded by this collaborative effort have allowed for the accurate diagnosis of what is likely an underreported disease in the Afghan population. This study demonstrates that collaboration can facilitate access to resources, such as immunohistochemical stains, which increases diagnostic accuracy.Future goals of this project include evaluation of treatment modalities, patient follow-up to assess disease status, and documentation of co-morbidities. This study is limited due to the paucity of cases and the referral bias of the patients. It is unknown whether individuals from represented provinces are more able to make the arduous journey for a more-advanced level of care. Alternatively, it is possible that there is a higher incidence of KS in these provinces. It is likely that these patients represent a small subset of KS cases in Afghanistan.This case series contributes valuable information to the body of knowledge for KS in Afghanistan, with the hope that this will increase awareness and, ultimately, improve outcomes for this disease.


Given the molecular structure and bulkiness of the diamine motor, we study the supramolecular assembly of the COF layers and detail stacking disorders between adjacent layers. We finally probe the motor dynamics with in situ spectroscopic techniques revealing current limitations in the analysis of these new materials and derive important analysis and design criteria as well as synthetic access to new generations of motorized porous framework materials.The incorporation of molecular machines into the backbone of porous framework structures will facilitate nano actuation, enhanced molecular transport, and other out-of-equilibrium host–guest phenomena in well-defined 3D solid materials. In this work, we detail the synthesis of a diamine-based light-driven molecular motor and its incorporation into a series of imine-based polymers and covalent organic frameworks (COF). We study structural and dynamic properties of the molecular building blocks and derived self-assembled solids with a series of spectroscopic, diffraction, and theoretical methods. Using an acid-catalyzed synthesis approach, we are able to obtain the first crystalline 2D COF with stacked hexagonal layers that contains 20 mol% molecular motors. The COF features a specific pore volume and surface area of up to 0.45 cm The synthesis of a light-driven molecular motor and its incorporation into a series of imine-based polymers and covalent organic frameworks is presented and criteria for the spectroscopic probing of molecular dynamics in porous solids are discussed. These include the ratio of dynamically active and passive components in confined space, the three-dimensional structure, adaptivity of the matrix and sufficient void space.Light-driven molecular motors have emerged as promising platforms to achieve stimuli-responsive motion at a molecular scale.17 and catenanes embedded in self-assembled monolayers18–20 and polymers21 result in a significant decrease in the rate of rotational and translational motion. Similar effects were observed for the intramolecular rotational motion of molecular motors embedded in polymers7,22 or grafted onto surfaces.23,24 Strategies to avoid these effects include incorporation into low-viscous fluids25,26 as well as spatial separation by co-assembly or immobilization of molecular motors into the backbone of porous frameworks8 and polymers.27 In particular, the latter is a critical innovation towards the transfer of controlled motion of a molecular machine onto a guest species.28 The Feringa group recently demonstrated that light driven molecular motors can be embedded in the backbone of metal–organic frameworks by applying a motor-based stator unit.8 The rotational frequency of the molecular motors in these nanoporous crystals did not change compared to the molecules' behavior in solution, indicating that the rotor units perform unrestricted motion. In principle, the pore space allows for dynamic confinement effects and unusual transport or adsorption phenomena.29 Castiglioni and Feringa et al. demonstrated that the porosity of a nanoporous polymer with backbone-embedded, light-driven molecular motors changes reversibly upon irradiation as a result of photoswitch dynamics.27 In a computational study, Kolodzeiski et al. predicted collective dynamics of framework-embedded molecular motors30 and Evans et al. were able to show that such properties can lead to activated, directed transport of a confined fluid mimicking a macroscopic pump.31 A critical design criterion of such activated directed transport is the presence of pore channels in which the fluid is confined and translational motion is forced in a preferred direction.High packing densities of bistable rotaxanes32 The unique properties, such as high porosity, large surface areas, light-absorption, and ordered structuring, are combined within a crystalline polymeric material. COFs have been used for diverse applications, including gas storage and separation, sensing, electrochemical energy storage, and heterogeneous (photo)catalysis.33–39A class of materials that feature such intrinsic pore channels and combines it with exceptional structural tunability are 2D Covalent Organic Frameworks (COFs).e.g. pore-accessibility, by external stimulation. To this end, various polymers40 and COFs comprising azobenzene-41,42 or dithienylethene-switches43 as building blocks have been synthesized. Apart from these switches, rotaxanes have also recently been introduced into COF-like polymers.44,45 A major challenge in these structures is the compromise between structural complexity, functionality and crystallinity of the material,46 with a loss of structural order evident in the X-ray diffraction pattern and low surface area with geometrically more complex building blocks.44,45 In contrast to MOFs, COFs are connected entirely by covalent bonds, and many COFs exhibit a higher chemical stability than MOFs.47 The combination of low density, structural tunability and stability makes COFs more attractive for the integration of molecular machines. As an example, many MOF structures are sensitive to solvent removal, drastically limiting their potential for applications.48 COFs furthermore perform better in terms of processability and in principle allow for hierarchical structuring, such as thin-film fabrication, required for advanced (photo-)electrochemical applications.49,50 Despite its structural elegance and advantages, molecular motors have not been embedded in COFs to date.Recently the incorporation of light-responsive moieties in COFs has gained interest for modifying their properties, 8 The spatial arrangement of molecular machines is crucial to amplify and coordinate their movement across multiple length and time scales.12 Molecular motors arranged in amphiphiles could be used as artificial muscles, able to lift macroscopic objects.51 Motors as dopants in liquid crystals, for example, are able to move micrometer sized objects through spatial rearrangement of the liquid crystal matrix.26 In these systems only small amounts of molecular motors were embedded, demonstrating that the quantity of machines integrated is less relevant than the quality of their arrangement to obtain responsive function.Contrary to amorphous polymeric materials, COFs allow, in principle, for precise arrangement of responsive molecules within their crystalline framework, similar to the arrangement demonstrated in MOFs.in situ spectroscopic methods. By utilizing different reaction conditions, we synthesize amorphous polymers as well as crystalline 2D COFs, constructed from this diamine molecular motor via imine condensation. The structural features of the self-assembled frameworks with respect to composition, local and long-range structure, structural disorder, and porosity were investigated. We elucidate the stacking mechanism of adjacent 2D layers in the framework and examine the motor behavior with FT-IR, Raman and UV/vis spectroscopy. From this, we derive design principles and methodologies that in the future will help to establish dynamic COFs to be used as light-responsive smart materials.Herein we detail design principles and synthetic strategies of COFs featuring backbone-embedded light-driven molecular motors. To the best of our knowledge, this is the first report of a light-driven molecular motor covalently incorporated in 2D COFs, in which both crystallinity and porosity are preserved. We synthesize a new diamine light driven molecular motor and investigate its dynamics with 52,53 First, the C–C double bond connecting the two halves of the molecule functions as the rotary axle. It undergoes an E–Z isomerization upon irradiation with light in the UV/vis region. The molecule adopts a metastable state, in which the methyl group of the upper half  as a suitable building block for the synthesis of COFs and 1,3,5-triformyl benzene (3) as the aldehyde counterpart  spectroscopy . Leaving the sample at 10 °C without irradiation led to recovery of the initial spectrum of the stable state  in mesitylene : 1,4-dioxane (1 : 4) at room temperature led to fast precipitation of an amorphous, polymeric material. Three different polymers with varying amount of motor were prepared: a pure motor polymer (m100-P), in a 1 : 1 ratio with the spacer (m50-P), and a pure spacer polymer (m0-P). After the condensation, the materials were washed with ethanol and centrifuged several times to remove residual building blocks and dried with supercritical CO2. The formation of the imine backbone of the polymer and the consumption of trisaldehyde 3 and amine building blocks 1 and 4 is confirmed by Fourier transform infrared (FT-IR) spectroscopy, showing the characteristic imine band  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>NC) at 1623 cm−1  under either of two reaction conditions. A Sc(OTf)1 Fig. S4. All thr13C ssNMR spectrum of m0-P shows three characteristic signals for the imine carbon atom (156.4 ppm), the aliphatic bridging atom (48.3 ppm) and the methyl groups (28.3 ppm) , indicative of only weak structural coherence within the material  and 10.2 m2 g−1 (m50-P) for the motor containing polymers, and moderate values of 713 m2 g−1 for the pure-spacer polymer m0-P, probed by N2 sorption analysis at 77 K  was performed to confirm the incorporation of the building blocks and preservation of motor integrity within the polymer backbone. The 69 With this approach m20-COF was crystallized from motor 1 and spacer 4 amine building blocks (4 : 1 molar ratio) and 1,3,5-triformyl benzene (3) in mesitylene as the solvent at 110 °C for 72 h . This supports the in-plane order of the layers, in spite of the random motor distribution. A Pawley refinement gives unit cell parameters a = b = 42.8 Å  stretching bands in the FT-IR spectrum and the presence of the imine bands (νNC) at 1623 cm−1 indicate condensation of the framework as previously described for the amorphous polymer  ssNMR spectra support this finding as well, showing a characteristic signal for the imine carbon at δ = 154.6 ppm (1H/13C heteronuclear correlation (HETCOR) spectroscopy  and the motor unit  incorporated into the framework. 13C direct-excitation (DE) experiments , and 17.8 ppm (motor) in the 13C-DE spectrum were found to be sufficiently resolved (l] as evident from transmission electron microscopy (TEM) analysis  and an average pore size centered at 2.7 nm was observed, derived by the QSDFT model for cylindrical pores  analysis.74The absence of amine  indicative of intralayer pore–pore correlations. Unlike other 2D COFs, interlayer correlations are not observed, indicating the relationship between the stacked, and even neighboring layers is not coherent, due to a broadened distribution of interatomic distances between neighboring layers.73,82 Hence, a good qualitative agreement to the simulated pair distribution functions (AA-1_EE model) is obtained by implementing large atomic displacement parameters in the [001] direction (U33), simulating different levels of reduction in interlayer coherence  and associated correlations of interlayer density.83 These are illustrated for the associated layered honeycomb structures in U11, U22) have been applied to wash out specific atom-pair correlations, resulting in effectively continuous density honeycombs. A shift from eclipsed to slipped stacking, for example, results in a broadening and effective shift of the interlayer density distribution , while a staggered relationship decreases the amount of nearest neighbor correlations but maintains the second neighbor correlations still sitting in an eclipsed position. With completely randomized layer offsets, the intensity of the interlayer peaks for the model are substantially diminished, but still present due to the remaining equidistant spacing and thus cannot explain the absence of interlayer correlations as experimentally observed for m20-COF. However, in the presented material conformational distortions are expected to be coupled with offsets, thus allowing the atoms in the layers to also move off their ideal positions in the stacking direction, resulting in a distribution of different interlayer distances. The combination of both offsets and conformational flexibility can lead to the extinction of the interlayer coherence, and no modulation in stacking density remains. This is further supported by simulated diffraction patterns for m20-COF as a function of randomized layer offset magnitude  and interlayer displacement parameter using DIFFaX84  were prepared under identical synthetic conditions as spectroscopic reference materials for COFs without motor-units (m0-COF) and with reduced pore-occupancy. Notably, these materials showed essentially identical XRPD patterns and FT-IR spectra to m20-COF  drastically impact the formation of ordered layered COF materials. Nevertheless, the residual porosity and pore channels may in principle allow for movement of the rotor units and potential intra-framework dynamic processes.In situ Raman spectroscopy of the dried powdered materials under irradiation was carried out to investigate light-driven dynamics of the molecular motors in the polymers and COFs. In previous reports on metal–organic frameworks, this technique enabled the detection of light-stimulated formation of the metastable isomer as well as the thermal relaxation of the metastable state of the motor after photoexcitation.8,11 With a half-life of 10.3 min for the imine functionalized motor 2, the isomerization process is expected to be observable at ambient temperature as confirmed by the switching of the motor 2 in toluene  sensitivity of analytical techniques towards low concentration of light-responsive functionality, and (ii) local and long-range structural disorder induced by the interaction of adjacent layers.85In a 2D COF, curved building blocks such as motor 1 and spacer 4 affect the self-assembled structure drastically. The curvature results in different conformational combinations, which reduce the average periodicity of the stacked layers. The conformation of the imine linkage as well as the steric demand of perpendicularly oriented methyl groups or rotor parts in spacer 4 and motor 1, respectively affect the stacking additionally. This has detrimental influence on interlayer stacking correlations and the available pore volume as well as the local environment of the motor. Yet, the accessible pore size is still found to exceed the radius of motor rotation. However, intermolecular interactions from adjacent layers may hamper or restrict motor movement even if commonly applied benchmarks for accessible void indicate otherwise. We thus conclude that local structural characteristics, such as interlayer interactions and stacking offset, which are invisible to most employed analytical methods, need to be considered for the development of analytical tools and the material's design to establish and investigate unrestricted motor dynamics in porous layered solids. Integrating molecular motors into three-dimensional COFs could circumvent this challenge. These materials are however challenging to synthesize and are prone to form interpenetrated structures, which can again constrain the available free volume for motor rotation.100-P). Our study thus highlights that new analytical techniques with high local sensitivity and in situ operation have to be developed to study the behavior of motor dynamics in this class of porous materials further. Advanced in situ ssNMR techniques under irradiation with abundant heteroatoms like fluorine and also 13C enriched motor units may enable to observe the rotation of embedded motors in these materials – if considered with structural characteristics, e.g. interlayer interactions and stacking offset, arising from the stacked layers. We envision that such analytical tools will also allow studies of interlayer dynamics and dynamic host–guest properties caused by the light-responsive dynamics of framework-embedded molecular motors. The synthetic procedures and in-depth structural characterization demonstrated herein provide access to these materials as model compounds and structural model systems. Furthermore, these can be used for in depth molecular dynamics simulations to probe design criteria for the operation of light-driven molecular motors in porous solids.Beyond these structural influences and design criteria, the commonly available and applied analytical techniques including solid state NMR, FT-IR, and Raman spectroscopy did not allow us to draw a definite conclusion on the rotation ability of the motor in the solid polymer and COF materials. In the presented COF materials the motor signals could not be detected due to the low concentration in the solid. An increased motor content, however, concomitantly reduces the available pore void and thus interferes with the free rotation of the motor units, as demonstrated with the motor-containing polymer (mhttps://darus.uni-stuttgart.de/) and are accessible upon request.Further data are stored on the DaRUS data repository (C. S. and L. G. contributed equally to this work. C. S. synthesized and analyzed the light-responsive molecular motors, imines, amines and derived polymers. L. G. synthesized and characterized the COF materials and established their structural models. M. W. T. and M. E. performed PDF analysis. W. R. B. performed Raman analysis. D. D. performed DFT analysis of the molecular motors. M. K. supported synthesis and spectroscopic analysis of the molecular motors. B. V. L., B. L. F. and S. K. supervised the project and supported writing of the manuscript. All Authors read and commented on the manuscript.The authors declare no competing financial interests.SC-013-D2SC02282F-s001