The uncoated and [p(4VP-co-EGDMA)-PVA] coated PVA-RB nanofiber mats were studied at different pH values to analyze their degradation and drug release profiles. The coated nanofibers demonstrated high stability at neutral and basic pH values for long incubation durations of 72 h, whereas the uncoated nanofibers dissolved in <2 h. The drug release studies showed that the RB release from coated PVA-RB nanofibers was higher at neutral and basic pH values, and proportional to the pH of the solution, whereas the degradation and RB release rates from the uncoated PVA-RB nanofibers were significantly higher and did not depend on the pH of environment. Further analysis of the release kinetics using the Peppas model showed that while polymer swelling and dissolution were the dominant mechanisms for the uncoated nanofibers, for the coated nanofibers, Fickian diffusion was the dominant release mechanism. The biocompatibility and therapeutic efficiency of the coated PVA-RB nanofibers against brain cancer was investigated on glioblastoma multiforme cancer cells (U87MG). The coated PVA nanofibers were observed to be highly biocompatible, and they significantly stimulated the ROS production in cells, increasing apoptosis. These promising results confirmed the therapeutic activity of the coated PVA-RB nanofibers on brain cancer cells, and encouraged their further evaluation as drug carrier structures in brain cancer treatment.Functional and stimuli-responsive nanofibers with an enhanced surface area/volume ratio provide controlled and triggered drug release with higher efficacy. In this study, chemotherapeutic agent Rose Bengal (RB) -loaded water-soluble polyvinyl alcohol (PVA) nanofibers were synthesized by using the electrospinning method. A thin layer of poly(4-vinylpyridine- Implantable patches for drug delivery have gained attention in recent years. In particular, these patches are used in site-specific, targeted delivery with enhanced sustained release of drug molecules from biodegradable materials  because they have high loading and encapsulation efficiency and they can be easily produced in a cost-effective manner –poly(L-lactic acid) (PEG–PLLA) diblock copolymer fibers loaded with an antineoplastic drug BCNU -1-nitrosourea) were fabricated to obtain controllable drug delivery directly to the tumor microenvironment  [p(4VP-co-EGDMA)], a pH-sensitive polymer, via initiated chemical vapor deposition (iCVD). iCVD is an all-dry, free-radical polymerization method that allows polymerization directly on the substrate surface, initiated by thermally decomposed radicals reacting with the monomer molecules adsorbed on the substrate  as a chemotherapeutic drug was reported. PVA polymer with RB solution was electrospun to form blend fibers. PVA is used as the polymer matrix due to its biocompatibility and biodegradable nature, leading to its wide utilization in drug delivery applications -coated PVA-RB nanofibers was investigated at different pH values, and the effect of the pH-responsive polymer coating on the release performance was studied. The degradation profiles of the nanofibers at these pH values were investigated to reveal their stability in cellular environment for their potential drug carrier activity. Moreover, the time-dependent anti-cancer activity of the coated PVA-RB nanofibers was studied on glioblastoma multiforme brain cancer cells (U87MG). The effects on cell viability were investigated as a preliminary study before exploring the therapeutic effect of the fibers via DNA quantification by using Pico Green dye. The intracellular RB localization and conformation of the cytoskeleton structure of cells was monitored by using confocal microscopy. The intracellular stress and death mechanism of the cells were investigated to analyze their cellular response against RB exposure. The significantly decreased cell viability and increased intracellular ROS production confirmed the increased apoptosis.In the study reported here, RB release from the p and Tserkovsky Bengal sodium salt  were used in electrospinning for fiber synthesis. The monomer 4VP , the cross-linker EGDMA , and the initiator tertbutyl peroxide  were used without purification. Phosphate-buffered saline  was utilized for the release studies.Ten wt% PVA solution was obtained by dissolving PVA in distilled water at 70°C and stirred continuously for 4 h. Homogeneous solution was observed after stirring overnight at room temperature, at 500 rpm. PVA-RB solution was prepared by blending 100 mg/ml RB distilled water and 10 wt% PVA solutions in RB:PVA 1:5 ratio. The loading capacity of the nanofibers was calculated to be ~16.7%.Electrospinning setup included syringe pump, stainless steel spinneret needle, high voltage supply, 10 × 10 cm collector, and 2 ml syringe. The distance between the needle and collector was kept at 15 cm, and the voltage was applied at 8 kV. Flow rates for both PVA and PVA-RB blend solutions were adjusted as 0.3 ml/h.co-EGDMA) was conformally deposited on both sides of nanofiber mats at a thickness of ~70 nm by using iCVD. The cross-linker EGDMA was heated to 95°C in a metal jar, and 4VP was kept at room temperature. The initiator was delivered to the system at a flow rate of 1 sccm while the flow rate of N2 gas was 1.1 sccm. The filament and substrate temperatures were at 240 and 25°C, respectively, throughout the deposition. Reaction pressure was fixed at 600 mTorr. Flow rates of 4VP and EGDMA were 2.73 and 0.14 sccm, respectively. Si wafers were coated simultaneously as the fiber mats, to be used in ellipsometric swelling experiments. P-coated PVA, and p(4VP-co-EGDMA)-coated PVA-RB nanofibers were imaged by field emission scanning electron microscopy  with an accelerating voltage of 4 kV. Chemical characterization of the fibers was performed by Fourier transform infrared spectroscopy  with 62 scans and the resolution of 4 cmXPS analysis was done by using Thermo Scientific K-Alpha spectrometer by an aluminum anode  at an electron take-off angle of 90° . The spectra were recorded using an Avantage 5.9 data system. The binding energy scale was calibrated by assigning the C1s signal at 284.5 eV.t – t0)*100/t0, where t is the thickness of the swollen and t0 is the thickness of the dry samples.Swelling behavior of the iCVD polymer film on Si wafer was investigated via a spectroscopic ellipsometer (M2000D J.A. Woollam Co. Inc.) in a liquid cell stage at room temperature at pH values of 4, 6.5, and 9. Dynamic thickness measurements were performed at 75° nominal angle of incidence within the wavelength range of 315–718 nm for 30 min. Swelling percentages were calculated by using the formula  following 24 and 72 h incubation times. The morphology of the fibers was visualized by monitoring the samples using SEM . Samples were incubated in 2-ml PBS solutions adjusted at pH 4, 6.5, and 9 for 24 and 72 h by shaking at room temperature. Following the incubations, the samples were dried under vacuum conditions for 24 h and prepared for SEM imaging. SEM imaging was carried out on samples gold-sputtered for 25 s at 60 nA, obtaining a 3 nm-thick conductive layer over the nanofibers.Coated and uncoated electrospun nanofibers were cut into 1 × 2 cm pieces and put in 10 ml PBS solutions separately at pH 4, 6.5, and 9 and placed on a shaker for the release experiments. UV-Vis measurements were performed using NanoDrop UV-Vis spectrophotometer (Thermo Scientific NanoDrop 2000c). For each measurement, 100 μl of PBS solution was removed from the solution and replaced with fresh PBS solution in the release medium. For RB detection, the UV absorbance peak at 550 nm was used. For each release experiment, eight sets of new samples were prepared and UV-Vis measurements were repeated at least twice on each sample.The amount of RB loaded in the nanofibers was determined by dissolving the uncoated fibers completely and measuring the RB concentration of the solution. Release percentages of the coated nanofibers were obtained by normalizing the concentration of RB released from the nanofibers by the amount loaded.2 atmosphere. Experiments were performed by seeding 30,000 U87MG cells/cm2 in 24-well multiwell polystyrene (PS) plates and by incubating the cultures with 0.8 × 0.8 cm2 coated PVA and coated PVA-RB nanofiber samples. For immunocytochemistry, cells were seeded on 0.8 × 0.8 cm2 Ibidi films made hydrophilic by O2 plasma  in a Colibrì reactor (Gambetti). Cells were exposed to nanofiber samples after overnight incubation.U87-MG tumor cells (from ATCC) were cultured under high glucose Dulbecco's Modified Eagle's Medium, added with 10% fetal bovine serum, 2 mM L-glutamine, and 100 U/ml penicillin−100 μg/ml streptomycin  in saturated humidity, 5% COex = 485 nm; λem = 535 nm).Cell proliferation was investigated by Pico Green assay (from Invitrogen) at 24 and 72 h from incubation with coated PVA and coated PVA-RB nanofiber samples. The assay enables ds-DNA quantification in solution by fluorescence measurement and requires cell lysis in a fixed volume of water (500 μl) by three cycles of freezing and thawing. To the purpose, 100 μl of working solution was mixed to 50 μl of cell lysate and to 150 μl of solution containing the PicoGreen dye. Samples were incubated in the dark for 10 min, and finally fluorescence was read with a microplate reader  and the total number of nuclei (m) were semi-automatically counted with ImageJ software and the n/m ratio (expressed as average ± standard deviation) was calculated and plotted. For statistical reasons, 10 images at low magnification were analyzed.U87MG cells were washed with PBS with calcium and magnesium, and fixed with 4% paraformaldehyde (from Sigma-Aldrich) in PBS for 20 min at 4°C. Then, cells were again rinsed with PBS and permeabilized with 0.1% Triton X-100 (from Sigma-Aldrich) in PBS for 30 min. Specific binding sites were saturated with 10% goat serum  in PBS for 1 h at 37°C. Then, cells were incubated with a 10% GS solution containing a rabbit polyclonal IgG primary antibody against Ki-67  for 3 h at 37°C. Samples were then rinsed four times with 10% GS (5 min each rinse), and cells were incubated with a 10% GS solution containing a goat polyclonal IgG secondary antibody  and 1 μM DAPI for 45 min at 37°C. After one rinse with 0.45 M NaCl PBS and with PBS, samples underwent fluorescence imaging. The number of Ki-67 immunopositive nuclei  by incubating 100 μl of cell suspension in 1× annexin-binding buffer, added with 5 μl of FITC Annexin V solution and with 1 μl of 100 μg/ml of propidium iodide. Staining was performed for 15 min in the dark. Flow cytometry was conducted with CytoFLEX platform .To quantitatively assess oxidative stress, cell cultures were stained with CellROX Green reagent (from Invitrogen) at 24 and 72 h from incubation with coated PVA and coated PVA-RB nanofiber samples. To the purpose, cells were rinsed with PBS without calcium and magnesium, and suspended by 1 min incubation with 0.05% trypsin (from Sigma-Aldrich). Cell pellets were obtained with 7 min centrifugation at 1,000 t-test. The data are expressed as the mean ± standard deviation (SD). Comparison with a p-value below 0.01 and 0.05 was considered statistically significant.Each experiment was carried out at least three times. Statistical analysis was carried out using an unpaired Student's SEM images of (a) uncoated PVA-RB nanofibers and (b) coated PVA-RB nanofibers are shown in co-EGDMA) coated PVA-RB nanofibers.As confirmed by the SEM images, the structures of the nanofibers were not damaged after the iCVD process and the fibers could be conformally coated with the polymer layer, enabling the fabrication of p(4VP-co-EGDMA) thin films on Si wafers was determined to be 62 ± 10% at pH 4, whereas at both pH 6.5 and pH 9, swelling was <5%, confirming the pH response of the polymer coating. The mesh sizes of the polymer coatings at pH values of 4, 6.5, and 9 were calculated as 1.16, 0.69, and 0.69 nm, respectively (Swelling percentage of p(4VP-ectively . Swellinectively .co-EGDMA)-coated PVA-RB nanofibers are shown in -1 are related to the C = O double bond, CH2, and C–O–C stretching modes of pure PVA nanofibers, respectively  was used to determine the surface composition of the PVA-RB nanofibers within 5–10 nm from the surface. co-EGDMA) coating on the nanofiber surfaces. The low percentages can be attributed to the fact that the coatings cover mostly the nanofibers, which are closer to the surface of the mat, leaving the nanofibers below the surface uncoated. Therefore, XPS analysis confirms the presence of a polymer coating on the nanofibers exposed to the surface.According to the XPS spectrum of the polymer-coated nanofibers, depicted in The degradation behaviors of coated PVA and coated PVA-RB nanofibers were investigated at several pH values  of PBS at increasing incubation times, as shown by SEM images . Uncoateco-EGDMA) coatings on the PVA nanofibers , which has a pKa in the range 4.5–4.7, occurs at lower pH values, leading to an electrostatic interaction between the protonated pyridine groups and the RB molecules. This attractive interaction also contributes to the reduced release percentages observed at pH 4.A transient behavior is observed at pH 6.5 with a faster early time release kinetics due to the collapsed polymer coating on the fibers, resulting in larger free space and shorter diffusion lengths for the dye molecules. However, overall release percentages at the end of 12 h are comparable to the release percentages at pH 4, but lower than the values obtained at pH 9.The early time release kinetics of the polymer-coated and uncoated PVA-RB fibers were investigated using the semi-empirical Peppas model, which includes relaxation and phase changes of the polymer matrix in addition to the diffusion of the drug molecules. The Peppas model in its simplest form is given by Korsmeyer et al. :Mt is the amount of drug released at time t, M∞ is total amount of drug loaded, “a” is a constant that depends on the structure and geometry of the of drug-polymer system, and “n” is the coefficient related to the mechanism of drug release  polymer was coated on PVA-RB blend nanofibers to attain pH response to the fibers and to tune the release kinetics. The vapor-based iCVD technique was successfully employed for the conformal deposition of the polymer coating without damaging the nanofiber mat. The coating layer enabled the control of the degradation and release kinetics of the nanofibers by tuning the pH of the medium. The iCVD was demonstrated to be a successful technique to coat the nanofibers with stimuli-responsive, functional polymer films to attain additional properties to the fibers.A pH-responsive nanofiber mat loaded with RB was produced as a potential controlled drug delivery system for post-operational cancer treatments. A thin layer of pH-responsive cross-linked p(4VP-The therapeutic efficiency of coated PVA-RB nanofibers against brain cancer was investigated comparatively against the coated PVA nanofibers, to analyze the anti-cancer effect of RB released from the fibers. The results indicated that coated PVA-RB nanofibers selectively decreased cell proliferation and stimulated cell death in apoptotic direction by increasing ROS production in the cells at long-term incubation as desired in chemotherapeutic applications.All datasets generated for this study are included in the article/in vitro cell culture experiments. GO and GC guided the study, wrote, and revised the manuscript.SSa designed the experiments, prepared the samples, and wrote the manuscript. AT performed the iCVD coatings and contributed to data analysis and interpretation. EO and SSh contributed to the electrospinning process and performed the release studies. ME, GG, and OS performed the drug release studies of the nanofibers with 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 reviewer CT-T declared a past collaboration with one of the authors GC to the handling editor.


In other words, what limits vs. promotes neurocognitive change as a result of second language acquisition in adulthood?Many individuals attempt to learn a second (L2) or even third language (L3) at some point in their life. Since language exposure is one of the most intense cognitive training regimes one can encounter, it is not surprising that previous research has shown that multilingualism can induce profound neural changes or “neuroplasticity”  acquisition. For instance, models of adult L2 learning assume that acquisition of the mother tongue (L1) has sculpted neural circuits to discriminate between L1 linguistic elements which in turn limits the ability to distinguish between L2 elements  both suggest that having reached adult age itself might be a limiting factor because adult age represents a period of relatively (as compared to childhood) low susceptibility to L2 exposure, limiting the degree to which L2 proficiency can be gained. Additionally, the mismatch of L1-L2 typology was suggested to limit L2 acquisition, with a relatively large mismatch delaying successful L2 acquisition (Antoniou and Wright). Indeed, a cross-linguistic priming study with concurrent ERP recordings presented in the current research topic showed that already early in the L2 acquisition process there is interaction between L1 and L2 at the semantic level . Another study , showed that a large typological difference between L1 and an L3 makes switching between languages in bilinguals more difficult with different underlying cognitive control networks being engaged in switching between balanced vs. unbalanced languages.What factors then, might limit neurocognitive change due to adult L2 acquisition? The two reviews presented in the current research topic .Acquisition of L2 (and underlying neurocognitive change) may additionally vary Yamamoto and Sakai), perhaps limiting acquisition of L2 syntax at adult age. On the other hand, the limiting effect of age of acquisition on L2 induced structural neuroplasticity may in itself be limited, as shown by a study presented in the current research topic, demonstrating white matter differences purportedly due to L2 learning between mono- and bilinguals, despite of L2 learners having reached adulthood .Finally, as outlined above (adult) age may itself limit neuroplastic change due to L2 learning. Indeed, a structural white matter imaging study presented in the current research topic suggests that white matter bundles critical for obtaining syntactic abilities are still developing in adolescence but may have reached maturation in adults (Birdsong) mentions some possible factors such as: high working memory capacity, motivation to learn and meta-linguistic awareness (which could be promoted by having successfully acquired a previous non-native language). Indeed, a study in the present research topic examining predictors of L2 acquisition success found evidence that high working memory capacity predicts L2 acquisition success . Furthermore, the general ability to learn or “language aptitude” may enhance neurocognitive change induced by L2 or L3 learning. Indeed, a study presented in our research topic investigating the morphology of Heschl's gyrus (HG), the primary auditory cortex, suggests that the number of complete duplications of HG in the right hemisphere might be a structural correlate of language aptitude, that may enhance L2 acquisition success . Finally, in an interesting study examining the effects of L2 acquisition on L1 processing presented in the current research topic (Kasparian and Steinhauer), very extended exposure to L2 and resulting high L2 proficiency emerged as an important factor in determining  morphosyntactic L1 processing, suggesting that the intensity of L2 exposure is a critical determinant of neuroplastic change in the underlying neurocognitive architecture of the language processing system.Having discussed the factors that may limit neuroplasticity due to adult L2 acquisition, what factors may promote it? One of the reviews of the present research topic  seems to be limited by adult age, typological mismatch between the already acquired and to be acquired languages, and limited exposure to the to be acquired language. On the other hand, high working memory capacity, high “language aptitude,” and a high level of exposure to the to be acquired language seem to promote neuroplastic change. Together, we have aimed with the studies presented in the current research topic to provide a fresh look at the scope of neuroplastic change due to adult second language acquisition.Conceived the idea: JW and NS; Wrote the Research Topic proposal: JW, YC, LP-R, MP, PW, and NS; Edited articles: JW, YC, LP-R, MP, PW, and NS; Wrote the editorial: JW.PW is co-owner of a tech startup company in Hong Kong that is related to this research topic. The remaining 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 fiber diameter and morphology of the nanofibrous DDSs were modulated by varying ultrasonic parameters in the USES process . We found that the USES technology produced nanofibers with higher fiber diameter (402 ± 127 nm) than TES (77 ± 21 nm). An increase of a burst count in USES increased the fiber diameter (555 ± 265 nm) and the variation in fiber size. The slight-to-moderate changes in a solid state  were detected when compared the nanofibers generated by TES and USES. In conclusion, USES provides a promising alternative for aqueous-based fabrication of nanofibrous DDSs for pharmaceutical and biomedical applications.We investigated nozzleless ultrasound-enhanced electrospinning (USES) as means to generate nanofibrous drug delivery systems (DDSs) for pharmaceutical and biomedical applications. Traditional electrospinning (TES) equipped with a conventional spinneret was used as a reference method. High-molecular polyethylene oxide (PEO) and chitosan were used as carrier polymers and theophylline anhydrate as a water-soluble model drug. The nanofibers were electrospun with the diluted mixture (7:3) of aqueous acetic acid (90%  Electrospinning (ES) is a method for fabricating polymeric nanofibrous constructs, which have potential applications in pharmaceutical and biomedical fields. Nanofibers are typically tenth-to-hundred nanometers thick, they feature large outer surface, substantial surface- area-to-volume ratio, and high porosity (nanomats). This makes these fibers interesting for drug delivery and tissue engineering applications ,2,3. To ES has been applied as a manufacturing method in the clothing, electronics and optical industries, and during the past twenty years it has gained increasing interest in the pharmaceutical and biomedical industries. In traditional ES (TES), a polymer solution is first translated via a capillary tube to a spinneret and then spun towards a grounded collector plate or roll using a high-voltage electron field between the spinneret and collector ,7. The mUltrasound-enhanced ES (USES) provides an orifice-free ES technique that employs ultrasound (US) to create nanofibers . In thisIn the present study, we compared the TES and USES techniques as means to fabricate drug-loaded polymeric nanofibers and we investigated the physicochemical and pharmaceutical properties of the produced nanofibers/nanofibrous DDSs. The influence of these two nanofabrication processes on the fiber formation, geometric fiber properties, surface morphology and physical solid-state properties of nanoconstructs were investigated. Special attention was paid to the formation and physical characterization of the drug-loaded nanofibers generated by the USES method.v/v) and formic acid  solution (90% v/v)  was used as a solvent system for ES.Theophylline anhydrate  was used as a water-soluble model drug. Polyethylene oxide, PEO  and chitosan   were investigated as carrier polymers in both TES and USES nanofabrication. The diluted mixture (7:3) of aqueous acetic acid   and in a custom-made in-house USES method. The USES method is described in detail in [The composition of the electrospun nanofibers is shown in etail in . In brieTo modulate the fiber diameter, specific US parameters  were exploited in an ES process. t-test) was made using Microsoft Excel 2016 .The nanofibrous samples were stored in a zip-lock plastic bag in ambient room temperature (22 ± 2 °C) prior to characterization. Scanning electron microscopy, SEM  and optical microscopy were applied to study fiber size distribution and morphology of nanofibers. The samples were coated with a platinum layer (6 nm) prior to imaging with SEM. Three SEM images of each sample were taken using three different magnifications . ImageJ software Version 1.51K was used to measure the diameter of nanofibers. Statistical evaluation  spectroscopy  with a single reflection attenuated total reflection (ATR) crystal , X-ray diffraction, XRD , and differential scanning calorimetry, DSC . XRD and FTIR spectroscopy results were normalized and scaled. In all DSC experiments, the sample size was 3–6 mg. The samples were first cooled down and kept at 0 °C for three minutes, and then heated to 350 °C at a rate of 10 °C/min. The samples were then cooled to 0 °C (10 °C/min) and then heated to 350 °C (10 °C/min). The DSC thermogram for PEO was obtained by heating the sample from 30 °C to 170 °C with a heating rate of 10 °C/min. For solid-state characterization, the corresponding binary or ternary physical mixtures (PMs) were prepared manually with a mortar and pestle, and they were used as reference samples for the nanofibrous samples.p< 0.001) was shown between the fiber diameter of nanofibers obtained with TES and the nanofibers generated with USES. This difference in fiber size could be explained by the fact that the USES is a multivariate process involving an open vessel and more critical process parameters (including US parameters) to be controlled than in the TES. The sensitivity of the polymer solution to US and the variations in distance between the surface of the ES solution and the collector plate could be potential reasons for these differences. In aqueous polymer solution ES, the process and ambient parameters such as conductivity, applied voltage, relative humidity, and the distance between a nozzle tip and collector plate could affect the diameter of nanofibers  [ fibers) . However fibers) .p < 0.001). In the TES, solutions with high conductivity and high surface tension require high voltages and the change in applied voltage (electric field) has only a minor effect on fiber diameter of the nanofibers [With USES, the fiber diameter can be modulated by changing the burst count (cycles per US pulse = duty factor). As seen in nofibers . Therefonofibers . As seenθ) at 19° and 23° [θ) at 7.2°, 12.6°, 14.3°, 24.1°, 25.6°, 26.4° and 29.4° [θ) is located at 12.6°. Crystalline chitosan has also two characteristic reflections (2θ) at approximately 10° and 20° [The carrier polymer PEO has two typical diffraction peaks  are absent in the XRD patterns of nanofibers generated by USES. Moreover, the other characteristic reflection (2θ) of theophylline at 12.8° 2θ is not distinguished in the XRD patterns of nanofibers (a new reflection shows less intensity and it is shifted to 13.5° 2θ). Therefore, it is evident that solid-state  changes in theophylline have taken place during the TES and USES nanofabrication. It is possible that in addition to an amorphous form, theophylline monohydrate or metastable theophylline or even the mixture of different forms may appear during or immediately after ES. Furthermore, all diffraction peaks in the XRD pattern of the nanofibers generated by USES appear weaker and less sharp than the corresponding reflections of the XRD pattern for the nanofibers produced by TES (θ) 19.2° which originates from PEO. With nanofibers generated by USES, the diffraction reflections (2θ) characteristic to semi-crystalline PEO at 19.2° and 23.3° are seen as slightly weaker than those in the XRD pattern of PM  of the pure materials, PM, and nanofibers produced by TES and USES are shown in 5 and 6. As seen in The DSC profiles of drug-loaded nanofibers generated by TES or USES were nearly identical suggesting that applying focused high-intensity US in the USES process does not significantly affect the solid-state properties of the nanofibers . As showIn the cooling phase, only the crystallization of PEO is observed in the DSC thermogram of the F-III nanofibers generated by USES . During −1 presents the stretching of C–H [−1 are derived from C–C and C–N bonds [−1) is partially visible in the FTIR spectra of the nanofibers produced by TES (F-I) and USES . The intensity of the characteristic peak of theophylline at 1658 cm–1 for nanofibrous samples , however, is smaller than that observed in the FTIR spectra of PM or pure theophylline powder.g of C–H . It can –N bonds . We founWe compared traditional electrospinning (TES) and nozzleless ultrasound-enhanced electrospinning (USES) as methods to fabricate nanofibrous polymeric drug delivery systems (DDSs). The physicochemical and pharmaceutical properties of the nanofibrous DDSs were studied. Both methods can be applied for aqueous-based fabrication of non-woven DDSs using PEO and chitosan as carrier polymers. With USES, the evaporation of solvent is advanced by using a high-intensity focused US enabling acoustic streaming and thermal effect inside the solution. Therefore, USES is associated with more pronounced process-induced solid-state changes of the materials compared to those induced by TES. Nanofibers generated by USES are amorphous, whereas the nanofibers produced by TES are less prone to being amorphous. The controlled phase transformation of higher-energy amorphous form is especially poorly applicable for water-soluble drugs. Further research is needed to discover all potential strengths and limitations of USES in fabricating nanofibrous DDSs.


Mw of (14.2 ± 1.2) kDa, and polydispersity index of ~ 1.2. Further analysis using MULTISIG revealed a distribution of material between 2 and 20 kDa and consistent with the weight average Mw. Controlled depolymerisation using hydrogen peroxide and ultra-violet radiation in an acetic acid medium reduced this to (4.9 ± 0.7) kDa, with a similar polydispersity. The depolymerised material appears to be within the range that has been predicted to fully penetrate into archaeological wood. The consequences for this finding and the use of the analytical ultracentrifuge in wood conservation strategies are considered.The heterogeneity and molecular weight of a chitosan of low molecular weight (molar mass) and low degree of acetylation (0.1) for potential use as a consolidant for decayed archaeological wood were examined by sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge before and after depolymerisation. Sedimentation velocity before depolymerisation revealed a uniform distribution of sedimentation coefficient with little concentration dependence. SEDFIT-MSTAR analysis revealed a weight average molecular weight  N-acetyl glucosamine)—has been considered for a wide range of applications from fining agents in the fruit juice industry, a condensation agent for DNA therapies, a mucoadhesive, an encapsulant, a film forming agent in shampoos and in wound healing technologies 2·12H2O . The consequence of this for application to alum treated wood was then considered.For the present study, we focussed on “Kitonor”  used by Christensen et al.  and usedKitonor chitosan was used  for 1 h at 20 °C. For UV exposure, the volume to surface area ratio was kept constant. The solution was then neutralized with 1 M sodium hydroxide. The precipitate was then centrifuged  at 10,000 revolutions per minute (rpm) and rinsed 3 times with 50 ml deionised H2O per vial. The solid product was frozen in a − 80.0 °C freezer overnight and freeze dried, resulting in a yield of 66–78%. The reaction was also carried out for 30 min and 90 min to investigate the effect of reaction time on Mw.Degradation of chitosan largely followed the procedure of Wang et al. . 4% chitῡ = 0.57 ml/g was used  was used and equipped with Rayleigh interference optics. 12 mm optical path double sector cells were employed: solution and solvent (buffer) reference channels were filled to 400 μl. A rotor speed of 50,000 rpm was used at a temperature of 20.0 °C. A 1.0 mg/ml stock chitosan solution in 0.2 M acetate buffer stock solution was prepared as above and then diluted to 0.80, 0.70, 0.60, 0.50, 0.45, 0.40 and 0.30 mg/ml. Analysis was carried out using SEDFIT  columns and a rotor speed of 35,000 rpm at a temperature of 20.0 °C. For the depolymerisation experiments, a higher rotor speed of 40,000 rpm  was employed. Analysis was carried out using SEDFIT-MSTAR .Table Mw ~ the mean Mw,app without the need for an extrapolation to c = 0. Mw = (14.2 ± 1.2) kDa.The molecular weights determined with the two lowest speeds  gave comparable data but at the highest speed  a significant reduction was found, a consequence of the loss from optical registration at the cell base of some the higher molecular weight part of the (polydisperse) distribution. This demonstrates that the popular practice used for protein work of using global analysis methods which average results over different speeds is unsuitable for highly polydisperse polysaccharides such as those being studied here. No significant change in molecular weight with concentration is observed Fig. , indicatM) vs M, MULTISIG analysis  kDa in exact agreement with SEDFIT-MSTAR. The analysis also yields an Mz = (16.4 ± 1.2) kDa giving a polydispersity Mz/Mw ~ 1.2.To estimate the distribution of molecular weights f kDa, a clear reduction in molecular weight compared with the native material of ~ 60%.The effects of depolymerisation treatment for 60 min on the molecular weight of the Kitonor chitosan are shown in Fig. w from MULTISIG = (5.2 ± 0.7) kDa, again in good agreement with SEDFIT-MSTAR, and much lower than the untreated sample. From MULTISIG, we also obtained an estimate for the z-average, Mz = (6.1 ± 0.5) kDa, giving a polydispersity Mz/Mw ~ 1.2, similar to the chitosan prior to depolymerisation, consistent with the higher molecular weights being depolymerised first  were investigated. The results from this are shown in Fig. Treatment with hydrogen peroxide coupled with UV light appears to degrade the chitosan in a controlled way. 30, 60 and 90 min reaction times show that the degree of depolymerisation can be appropriately chosen, at least for Kitonor chitosan. What is apparent from Fig. xw is just (Mw/mo) with mo the molecular weight of the repeat unit, Eq  h−1.We can estimate a depolymerisation decay constant k from the relation of Tanford (Eq. 33.12 of Tanford Mw = (5.35 ± 0.70) kDa  which proves the consistency of the results. Reproducibility can also be an issue with depolymerisation. However, keeping the same concentrations, reaction times and volume to surface area ratio, it is clearly possible to get similar molecular weights using the same starting material.The effect of “scale-up”—processing of larger quantities of material—was then investigated. 5 × 22 g batches of chitosan were each depolymerised for 1 h. Encouragingly these consistently gave the same molecular weight To increase the quantity of chitosan being depolymerised, an in-flow UV depolymerisation system could be set up but the reaction times may need to be adjusted to fit with the change in surface area exposure to UV light and volume.In this study, we have demonstrated that it is possible to produce chitosan of a suitable molecular weight range—which could form the basis for a consolidant for archaeological wood and in a controllable fashion. We have also established a procedure for determining the molecular weights and molecular weight ranges of consolidant materials on an absolute and matrix-free basis and for determining their stability against decay.We have investigated chitosan at one degree of acetylation DA = 0.1. Examination of chitosans of other DA’s may prove useful, although the stability of chitosans seems to be independent of DA (Holme et al. This paper was presented at 5th EPNOE International polysaccharide conference held in Jena, Germany, August 2017 and at the 23rd analytical ultracentrifugation conference held in Glasgow, UK, September 2017.


Antagonists of both D1- and D2-class dopamine receptors partly reduce the dopaminergic calcium response, indicating that both receptor classes contribute to dopamine-induced calcium transients in olfactory bulb astrocytes.It is well established that astrocytes respond to the major neurotransmitters glutamate and GABA with cytosolic calcium rises, whereas less is known about the effect of dopamine on astroglial cells. In the present study, we used confocal calcium imaging in mouse brain slices of the olfactory bulb, a brain region with a large population of dopaminergic neurons, to investigate calcium signaling evoked by dopamine in astrocytes. Our results show that application of dopamine leads to a dose-dependent cytosolic calcium rise in astrocytes (EC Besides that, dopamine is involved in diverse diseases, such as schizophrenia, Parkinson’s disease and depression. To accomplish all these functions, there are 11 populations of dopaminergic neurons distributed in the CNS2. Dopaminergic neurons in the olfactory bulb (OB) are the most numerous among these cell groups3 and are subdivided in two subgroups: the short axon (SA) cells and a subpopulation of external tufted (ET) cells4. Within the OB, dopaminergic neurons have been reported to be found mainly in the glomerular layer, which is populated by a variety of different periglomerular interneurons. 10–16% of them are supposed to be dopaminergic6. A common property of the OB dopaminergic neurons is the ability to co-release dopamine and GABA from separate vesicle pools9. Dopaminergic neurons are the only catecholaminergic neurons located in the OB, hence they could be recognized by the expression of tyrosine hydroxylase (TH), a rate-limiting enzyme in the biosynthesis of dopamine11. Dopamine acts on dopamine receptors that are metabotropic G protein-coupled receptors containing seven transmembrane domains. Based on coupling to either Gαs,olf proteins or Gαi/o proteins to stimulate or inhibit the production of the second messenger cAMP, respectively, dopamine receptors are classified as D1-class receptors (including D1R and D5R) or D2-class receptors 14.Dopamine (DA) is one of the most important modulatory neurotransmitters in the mammalian brain. It has several physiological functions in the central nervous system (CNS) including modulation of voluntary movement, reward, sleep regulation, feeding, affect, attention, cognitive function and olfaction1-class receptors in cultured astrocytes of different brain regions17. In addition to the studies above describing dopamine-induced calcium transients in cultured astrocytes, Jennings et al. (2017) and Xin et al. (2019) could demonstrate that hippocampal and midbrain astrocytes in acute brain slices respond to dopamine with a cytosolic calcium rise. However, the effect of dopamine on astrocytes in other brain regions such as the olfactory bulb remains unknown and has been investigated in the present study.Although the actions of dopamine on neurons are well described, its influence on astroglia remains poorly understood. There has been evidence for the presence of dopamine receptors leading to cAMP production and subsequent PKA activation as well as NADH modulation and calcium signaling mediated by D1-class and D2-class receptors.We intended to elucidate whether olfactory bulb astrocytes respond to dopamine with calcium signaling and to identify the receptor subtypes involved as well as the origin of the intracellular calcium. Our results show that astrocytes respond to bath application of dopamine with calcium release from internal stores by activation of both Dfl/fl mice and aimed to visualize astrocytes by anti-GFAP staining. GFAP-positive astrocytes were located throughout the GL, which can be distinguished from the ONL by glomerular structures, and the EPL, in which GFAP-expression was weaker compared to the GL  and bath application  led to transient elevation in cytosolic calcium in OB astrocytes  with the corresponding control application in the absence of receptor antagonists to assess the contribution of these receptors to the dopamine-evoked response  has also been described by both D1-class and D2-class dopamine receptors29. To ascertain whether the calcium rise depends on the PLC/IP3 signaling pathway, we applied dopamine in the presence of the IP3 receptor antagonist 2-APB  in amplitude and 71.3+/− 3.1%  in area, suggesting the involvement of IP3 receptors . Depletion of internal stores led to a long-lasting elevation in intracellular calcium and suppressed dopamine-evoked calcium responses. The mean amplitude of dopamine-induced calcium transient was reduced by 93.8+/− 2.5%  and the area by 88.2+/− 2.9%  Fig. , indicatAPB Fig. . A relators Fig. . In addiors Fig. . In ordeors Fig. . Interesors Fig. .Figure 41-class as well as D2-class dopaminergic receptor antagonists on dopamine-induced calcium transients. In the presence of the D1-antagonist SCH23390, the dopamine-induced calcium transient was reduced by 47.2+/− 5.0%  in amplitude and 64.1+/− 6.2%  in area  in amplitude and 48.2+/− 6.9%  in area . The most common mechanism to liberate calcium from the ER is the PLC/IP3 signaling pathway, which has previously been shown in OB astrocytes to be activated by P2Y1 receptors, A2A receptors and mGluR530. Although dopamine receptors are mainly considered to be linked to adenylate cyclases, several instances have shown stimulation of PLC by both D1-class and D2-class dopamine receptors . Hence, it is likely that dopamine releases calcium from the ER by stimulation of PLC. Based on this assumption, we performed experiments in the presence of phospholipase C inhibitor U73122 and the IP3 receptor antagonist 2-APB, which both resulted in a considerable decrease in dopamine-induced calcium rise. Furthermore, the kinetics of the calcium response was altered in the presence of both 2-APB and U73122. The property of 2-APB acting not only as an IP3 receptor antagonist, but also as an inhibitor of store-operated calcium entry (SOCE), may provide an explanation for the rapid decay of the calcium rise after reaching its peak, since inhibition of SOCE channels suppresses the late phase of the calcium response31. This result may be considered as a first indication of the involvement of SOCE in dopamine-induced calcium transients in astrocytes. In contrast, in the presence of the PLC inhibitor U73122, despite a significant reduction in mean amplitude, a prolongation of the dopamine-induced calcium transient could be observed. This might be due to the U73122-mediated attenuation of the PKC signaling pathway, which acts downstream of PLC activation as a negative feedback mechanism to terminate calcium elevations evoked by G protein-coupled receptors33. Our results suggest major contribution of the PLC/IP3 pathway to dopaminergic calcium responses in OB astrocytes, however, both U73122 and 2-APB failed to entirely block dopamine-induced calcium transients. We cannot exclude that the concentrations of the compounds at their targets were not sufficient to totally block PLC-mediated IP3 receptor activation, since U73122 does not easily diffuse within tissue and, in order to avoid activation of TRPV channels, the concentration of 2-APB could not be raised to values needed for complete block of IP3 receptors24.In the present study we showed that dopamine induced an increase in intracellular calcium concentration in olfactory bulb astrocytes 1- and D2-class receptor antagonists, thus both classes of dopamine receptors appear to contribute to the dopamine-evoked calcium transients. This is in line with observations suggesting that both D1- and D2-class receptors elicit release of IP3 and calcium signaling in astrocytes29. Whether this effect in OB astrocytes is mediated by D1-D2 heteromers, as shown in striatal neurons34, or by independent D1 and D2 receptors is not known and needs further investigation. In contrary to our results, studies in the hippocampus and ventral midbrain have shown that activation of D2 receptors leads to a decrease in cytosolic calcium, whereas D1 receptors mediate a calcium rise35. The D2-class receptor-mediated decrease in calcium in hippocampal astrocytes presumably results from modulation of L-type voltage-gated calcium channels29, which has also been shown in nucleus accumbens neurons36. Apparently, this mechanism does not exist in astrocytes of the olfactory bulb. However, it must me mentioned that, due to the limitations of two-dimensional confocal imaging and lack of visibility of small cell processes after bulk loading of calcium indicators, it is possible that other small-scaled but functionally important calcium signals in fine astrocyte processes may have been unexploited. Furthermore, in comparison to the work of Jennings et al.29, there are some considerable differences in experimental design, such as our method of application differed in terms of duration. While Jennings et al. used a puff application of 3 minutes and bath application of 10 minutes in most trials, it was sufficient to confine the dopamine application to 2 seconds (puff application) and 30 seconds (bath application) in our study, suggesting a high dopamine sensitivity in astrocytes of the OB, but only moderate sensitivity in the hippocampus. This is confirmed by the long delay in onset of the calcium response often seen in hippocampal astrocytes29. The dopamine sensitivity of OB astrocytes might even be underestimated, since bulk-loading of calcium indicators results in significantly smaller calcium-dependent fluorescence changes compared to loading single cells (and gap junction-coupled cells) via a patch pipette29.The dopamine-induced calcium elevation was decreased by both D10, the distribution of the D1-receptor is widely spread over all OB layers except of the nerve layer42. This indicates a functional role of dopamine in the entire OB, including the glomerular layer as well as in external plexiform layer, which is consistent with our results. The distribution of the D2 receptor has its highest density in the GL and nerve layer, while D2 receptor expression in the EPL is sparse45. Despite that, the dopamine-induced calcium transients where strongly reduced in the presence of D2 antagonist sulpiride in both the GL and EPL, indicating expression of D2 receptors in astrocytes of the EPL. Previous studies by Pignatelli et al.37 showed that during the process of maturation of adult-born dopaminergic neurons, two populations of TH+ neurons outside the glomerular layer exist. One of these populations represents dopaminergic neurons that undergo the last step in maturation and can be found in the mitral cell layer and external plexiform layer37. These cells project numerous processes within the EPL, suggesting release of dopamine not only in the GL but also in deeper layers. As shown in Fig. 46.Although the localization of tyrosine hydroxylase and thus dopamine-containing neuronal elements has its highest density in the glomerular layer Fig. 10, the d48. In the main olfactory bulb, several studies provided evidence for important functions of astrocytes, e.g. gliotransmission49. Kozlov et al. demonstrated that astrocytes in the rat olfactory bulb release two classical transmitters, GABA and glutamate, upon mechanical stimulation to evoke synchronous currents in mitral and granule cells50. Another gliotransmitter released by OB astrocytes is ATP that is degraded to adenosine, a potent neuromodulator in the OB51. In mitral cells, adenosine activates two-pore domain potassium channels and inhibits presynaptic calcium channels, resulting in reduced spontaneous firing and attenuated reciprocal dendro-dendritic inhibition53. OB astrocytes also play a key role in neurovascular coupling, since it has been shown that the physiological activation of olfactory sensory neurons by odors reliably triggers calcium increases in astrocyte processes in the glomerular layer and subsequent dilation of blood vessels54. Neurovascular coupling is not restricted to the GL, since astrocytes transmit panglial calcium waves to olfactory ensheathing glial cells in the nerve layer, which there contribute to regulation of blood vessel diameter56. Which role dopaminergic calcium signaling in astrocytes plays in neuronal performance and neurovascular coupling in the OB is not known so far and needs further investigation. However, tuning of olfactory information processing and odor perception by neuromodulators such as dopamine, noradrenaline, acetylcholine and serotonin has moved into the focus of research on olfaction, in particular in recent years59, and it is becoming increasingly clear that astrocytes have to be considered to take actively part in these processes. This hypothesis appears to be supported by findings showing interactions of dopamine and astrocytes in other regions of the CNS, such as ventral midbrain and hippocampus35.It is well known that astrocytes are involved in a variety of processes in the CNS. Apart from well-established supportive astrocytic functions such as metabolic supply and potassium homeostasis, more recent studies revealed that astrocytes are tightly integrated into neural networks in a functional manner. As a result of astroglial calcium excitability, astrocytes are capable of “gliotransmission”, i.e. release of transmitters which can lead to signaling to neighboring neurons and blood vessels2) and decapitated before using for experiments. The permission to despatch mice for the purpose of organ extraction was obtained by the Hamburg Authority of Health and Consumer Protection . According to the local laws, an additional ethical approval for animal experiments in the current study was not required. Olfactory bulb slices were prepared as described before60. Slices were quickly transferred into a chilled artificial cerebrospinal fluid . 200 µm thick horizontal slices of the bulbs were cut using a vibratome . Brain slices were stored in ACSF for 30 min at 30 °C and 15 min at room temperature before starting experiments. ACSF was continuously gassed with carbogen .Naval Medical Research Institute (NMRI) outbred mice from postnatal day 7 (p7) to p21 were used for calcium imaging experiments. The mice were bred in the institute’s animal facility at the University of Hamburg. This study was carried out in accordance with the recommendations of the European Union’s and local animal welfare guidelines. Mice were anesthetized using isoflurane  and ZM241385 (4-(2-triazolotriazin-5-ylamino]ethyl)phenol) was obtained from Tocris . D-APV (D-2-amino-5-phosphonopentanoic acid), NBQX , gabazine (6-Imino-3-(4-methoxyphenyl)-1(6 H)-pyridazinebutanoic acid hydrobromide), TTX -2-imino-5, 9:7, 10a-dimethano-10aH-dioxocino pyrimidine-4, 7, 10, 11, 12-pentol + citrate buffer), CPA -10-acetyl-2, 6, 6a, 7, 11a, 11b-hexahydro-11-hydroxy-7, 7-dimethyl-9H-pyrroloisoindoloindol-9-one, SCH23390 ((R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine) and sulpiride -(±)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxybenzamide) were obtained from Abcam . All substances were stored as stock solutions according to the manufacturers’ description.The standard ACSF for acute brain slices contained (in mM): NaCl 125, KCl 2.5, CaCl7630403G23RikTg(Th-cre)1Tmd/J) × tdTomatofl/fl mice (P28) was performed as described62. These mice express the red fluorescent reporter tdTomato under control of Cre recombinase, which is expressed only by cells with active tyrosine hydroxylase promoter. After dissection, the olfactory bulbs were kept for 1 h at room temperature (RT) in formalin . Subsequently, 120 μm thick sagittal slices were prepared with a vibratome  and incubated for 1 h in blocking solution  at RT. Afterwards, the slices were incubated for 48 h at 4 °C with a chicken anti-GFAP antibody . The antibody was diluted in 1% NGS, 0.05% Triton X-100 in PBS. Slices were incubated in PBS with the goat anti-chicken Alexa 488 secondary antibody  for 24 h at 4 °C. Subsequently, the slices were mounted on slides using self-hardening embedding medium . Immunohistological stainings were analyzed using a confocal microscope . Confocal images were deconvolved using a theoretical point-spread function  and enhanced for brightness and contrast using ImageJ and Adobe Photoshop CS2.Immunohistochemistry on olfactory bulbs of TH-Cre (B6.Cg-2/5% CO2). Bath perfusion with ACSF was accomplished using a peristaltic pump . Drugs were applied via the perfusion system. If not stated otherwise, ACSF contained 1 µM TTX to suppress neuronal activity. Changes in intracellular calcium levels in olfactory bulb astrocytes were recorded by confocal microscopy . An excitation laser wavelength of 488 nm and a frame rate of 0.75 fps were used and the fluorescence was collected through a 500–530 nm bandpass filter.Slices were incubated with the membrane-permeable form of the calcium indicator Fluo-8  made from a 2 mM stock solution (dissolved in DMSO and 20% pluronic acid) for 30 min. Brain slices were then placed in the recording chamber and fixed with a U-shaped platinum wire with nylon strings. Brain slices were continuously superfused at a rate of 2 ml/min with ACSF that was gassed with carbogen  was recorded during the course of the experiment and normalized to the basal fluorescence intensity in absence of pharmacological stimuli. Changes in calcium are given by ΔF as the percentage changes in fluorescence with respect to the basal fluorescence. Calcium responses were assessed as amplitude (baseline to peak) and area . All values are given as mean values (+/−) standard error of the mean with “n” representing the number of cells analyzed for a certain set of experiment. In the figures, sample size is given as “number of cells/number of brain slices/number of animals”. The proof of statistical significance was done by Wilcoxon signed-rank test at an error probability p . In case of independent data the Mann-Whitney-U test was applied. The dose-response curve was generated using the response to 100 µM dopamine, a concentration used in all measurements analyzed for the dose-response relationship, as reference. The values of all responses to 100 µM dopamine were averaged, resulting in 85.2+/− 5.1% ΔF, and ΔF values of all individual cells were normalized to this value. Normalized values were pooled and the dose-response curve was generated using Origin Pro 9.1.The data were evaluated with Nikon EZ-C1 Viewer (Nikon) and statistical tests have been applied with Origin Pro 9.1 . To analyze changes of the calcium level in astrocytes, cell somata were marked as regions of interests (ROIs). Cells located in the glomerular layer and external plexiform layer that showed a calcium response to ADP were identified as astrocytesSupplementary Information.Supplementary Information.


Pogonomyrmex barbatus) differ in how they regulate collective foraging activity in response to changes in humidity. We used transcriptomic, physiological, and pharmacological experiments to investigate the molecular basis of this ecologically important variation in collective behavior among colonies. RNA sequencing of forager brain tissue showed an association between colony foraging activity and differential expression of transcripts related to biogenic amine and neurohormonal metabolism and signaling. In field experiments, pharmacological increases in forager brain dopamine titer caused significant increases in foraging activity. Colonies that were naturally most sensitive to humidity were significantly more responsive to the stimulatory effect of exogenous dopamine. In addition, forager brain tissue significantly varied among colonies in biogenic amine content. Neurophysiological variation among colonies associated with individual forager sensitivity to humidity may reflect the heritable molecular variation on which natural selection acts to shape the collective regulation of foraging.Colonies of the red harvester ant (  •Red harvester ant colonies vary in how they adjust foraging effort in dry conditions•Colonies that differ in behavior significantly differ in forager brain transcriptome•Pharmacological increases of dopamine increased foraging in field experiments•Foragers from colonies more sensitive to humidity were more stimulated by dopamine Entomology; Neuroscience; Molecular Neuroscience Many biological systems, from brains to insect colonies, are regulated by distributed processes based on local interactions. Variation among groups in collective behavior  can arisAcross ant and bee species, variation among nestmates in reproductive status and behavioral performance are associated with tissue-specific physiological and transcriptomic differences . Worker Pogonomyrmex barbatus, forage in the desert for seeds that provide both food and water. Foragers lose water while out in the desert sun, and the rate of water loss is higher in dry conditions  to assess transcriptomic differences in forager brain tissue between 2 sets of colonies that naturally varied in how strongly they reduced foraging activity in dry conditions. Patterns of transcript differential- and co-expression between the 2 sets of colonies included significant changes in biogenic amine and neurohormonal pathways. To test the role of dopamine signaling in the regulation of individual foraging activity, we manipulated the brain dopamine titer of foragers in field experiments during 2 consecutive years. Foragers with increased brain dopamine titer made significantly more foraging trips than control-treated nestmates, and foragers treated with a metabolic inhibitor of dopamine synthesis significantly decreased foraging activity relative to control-treated nestmates. In the set of 9 colonies used in pharmacological experiments, we also characterized natural patterns of behavioral variation and forager brain biogenic amine content. Colonies that were naturally more sensitive to humidity tended to be more responsive to the stimulatory effect of exogenous dopamine. This suggests that a forager's decision whether to leave the nest on its next trip may be influenced by dopamine, so that variation among colonies in the regulation of foraging in response to conditions may be due to differences among colonies in forager biogenic amine neurophysiology.P. barbatus colonies that differed in how strongly they reduced foraging activity in dry conditions (see P. barbatus reference transcriptome (2 = 0.99). A linear principal component analysis in sleuth (Forager brain transcriptomes differed between 2 sets of ions see  1A. We cions see  to quantcriptome . Of the criptome A. A totan sleuth  showed t−7). The enrichment of the GO term “neuropeptide signaling pathway” was driven by increased expression of transcripts from pathways involved in the regulation of insect foraging behavior, including the FMRFamide receptor , an allatostatin peptide hormone , and the hypertrehalosaemic prohormone , all of which are important in insect neurohormonal signaling in solitary insects (−5), a phosphoinositide phospholipase , and the glycogen synthase kinase 3β interaction protein . These 3 protein products are involved in the inositol phosphate signaling pathway . The 160 transcripts upregulated in colonies that did not reduce foraging in dry conditions did not show any GO term enrichment with FDR < 0.9. The 113 transcripts upregulated in the colonies that strongly reduced foraging in dry conditions were enriched in GO terms “neuropeptide signaling pathway,” “catecholamine metabolic process,” and “receptor binding” . The enrichment in biogenic amine signaling and metabolism GO terms was reflected in the higher expression of the neurometabolic enzymes phenylalanine hydroxylase  and tyramine β-hydroxylase  treatment group were significantly increased by 2.67-fold relative to controls  on foraging activity. 3IY is a metabolic inhibitor that reduces brain dopamine titer in insects  however,Next we asked how variation in colony response to pharmacological manipulation was associated with natural variation in how strongly colonies reduced foraging activity in dry conditions. Colony reduction of foraging activity in response to humidity was quantified by estimating the decrease in daily foraging trips made by the colony per percent decrease in humidity see  1F. As i2 = 0.52, p = 0.028). There was no significant relation between a colony's response to 3IY and how strongly it reduced foraging activity in dry conditions (Pearson's correlation test p > 0.7).Colonies that more strongly reduced foraging activity in dry conditions were more responsive to the stimulatory effect of exogenous dopamine  .


This study aimed to investigate the morbidity profile and the sociodemographic characteristics of unaccompanied refugee minors (URM) arriving in the region of Bavaria, Germany, between October 2014 and February 2016.The retrospective cross sectional study included 154 unaccompanied refugee minors between 10 and 18 years of age. The data was derived from medical data records of their routine first medical examination in two paediatric practices and one collective housing for refugees in the region of Bavaria, Germany.Only 12.3% of all participants had no clinical finding at arrival. Main health findings were skin diseases (31.8%) and mental disorders (25%). In this cohort the hepatitis A immunity was 92.8%, but only 34.5% showed a constellation of immunity against hepatitis B. Suspect cases for tuberculosis were found in 5.8% of the URM. There were no HIV positive individuals in the cohort. Notably, 2 females were found to have undergone genital mutilations.The majority of arriving URM appear to have immediate health care needs, whereas the pathologies involved are mostly common entities that are generally known to the primary health care system in Germany. Outbreaks due to hepatitis A virus are unlikely since herd immunity can be assumed, while this population would benefit from hepatitis B vaccination due to low immunity and high risk of infection in crowded housing conditions. One key finding is the absence of common algorithms and guidelines in health care provision to URM. In the year 2015 there were 21.3 million people forced to leave their home countries worldwide. The majority of the refugees (54%) came from the following three countries: the Syrian Arab Republic with 4.9 million, Afghanistan with 2.7 million and Somalia with 1.1 million [In Germany the numbers of asylum seekers increased substantially in recent years, culminating in a peak influx of refugees into the German state of Bavaria towards the end of 2015. According to Eurostat the number of asylum applications by unaccompanied refugee minors (URM) in Germany more than quintupled (from 4400 to 22.255) from 2014 to 2015 , which iAccording to an assessment by the European Centre for Disease Prevention and Control the refugees are considered to be generally in a good health status at the beginning of their journey . HoweverSeveral recent studies from Germany and other countries receiving refugees in Europe have covered the prevalence of communicable and non-communicable diseases in arriving refugees, mostly in adult populations. Just a few studies on refugee minors have been published so far. Mockenhaupt et al. have found a prevalence of intestinal parasites of 22% in 488 Syrian URM in Berlin, whereas 66% were considered to be healthy in this cohort . In a stRecipient countries like Canada or Australia developed already years ago very detailed guidelines to support primary health care practitioners in an adequate handling with medical examinations of new arriving refugees –16. For For the current refugee health situation in Germany there is a growing wealth of data on disease prevalence in arriving refugee populations in general, however, it has to be mentioned that data on the morbidity profile in URM is scarce . In ordeThis is a retrospective cross-sectional study. Anonymized medical data from paper based files of newly arrived URM were analyzed. The data from the initial medical examination after arriving to Germany was collected in two specialized practices for paediatrics (location A and location B) and in a collective housing for refugees  in the state of Bavaria (South Germany). All examining physicians were specialized paediatricians.Data was collected from October 2014 until February 2016. The initial medical examination was done regardless of existing signs of illness as stipulated by Section 62 of the German Asylum Act. Hence the examinations were mandatory. All examinations were conducted under language barriers, since URM were generally not speaking any German. However interpreters were provided occasionally by the Youth Welfare Office, while in other cases fellow refugees were translating into English. The Youth Welfare Office was at the same time acting as the legal representative conveying consent to the medical interventions conducted.All three performing parties used their own self developed examination form to record the information. Therefore in each sub-cohort the range of collected variables is different.Information on sex, age, country of origin and the escape route (via sea or land) were collected. Since age was recorded in full years only, some URM that were 17 years of age but had their birthday that same year are classified as 18 years of age. All screening contacts were conducted under substantial time constraints by the executing physicians.All collected data were entered at the different sites onto paper-based data collection forms. A physical examination was performed. Blood samples were tested for hepatitis A, B and C, human immunodeficiency virus (HIV) and syphilis . In addition, a differential blood count was done. Stool diagnostics were performed and included an antigen test through an enzyme immunoassay for Helicobacter pylori (not performed in location C), amoeba, lamblia and microscopy for parasites.The URM underwent diagnostic tests for tuberculosis (Tb), either through an Interferone Gamma Release Assay (Quantiferon Gold Test) which was envisaged for URM below 16 years (only performed in location A and B). In URM of 16 years and above a chest X-Ray was the standard screening measure. However, chest X-Ray results are only available for location B, since adequate feedback mechanisms were missing in locations A and C. Testing for Tb was not performed in location C. In suspect cases a referral to a pulmonologist was ordered. In location A previous contact to known Tb patients was documented.3/μl and for thrombocyte counts 130–350 × 103/μl. The results for mean corpuscular volume (MCV) were coded as pathological or normal in all three locations. Erythrocytic microcytaemia was defined as MCV below defined threshold levels. The applied thresholds were different across locations. Location A and B had a lower limit of 76.7 fl, whereas in location C the threshold for URM below 17 years was 79 fl, and for 17 years and above 80 fl.In location A and B the resultsfor Eosinophilia were documented only as pathological or normal. The threshold for a pathological finding for Eosinophils was 4%. The range of normal for leucocyte counts was 4.0–10.0 × 10The thresholds for haemoglobin (Hb) were as well different across locations. In location A and B the results for Hb were documented only as pathological or normal. Location A and B had a lower limit of 11.0 g/dl, whereas in location C the threshold for URM below 16 years was 12.8 g/dl, and for 16 years and above 13.5 g/dl.The divergent thresholds for MCV and Hb had to be sustained in the course of the analysis, as data was only available in a dichotomous format as above or below threshold .A psycho-social questionnaire was applied to the URM at location A. This tool has been designed by the responsible physician based on personal experiences in psychosocial assessment of paediatric cases. The physician expressed a suspicion of post traumatic stress disorder (PTSD) based on his overall clinical judgement and presence of three key components of the questionnaire: sleeping disorders, nightmares and frequent headaches. In location B the responsible physician has also expressed suspicions of PTSD, however, the assessment algorithm was not found to be reproducible, the cases from location B are therefore not included. It has to be pointed out that the data on findings in location A are indicating an overall magnitude of distress in the target population as a whole, potentially needing structured intervention at later stages, rather than individual diagnoses.For the data analysis the data of all three locations were combined and analysed as a cohort at large as the different sites examined comparable target populations in the same geographical area.Data was entered into an Excel 2010 database. Data entry was double checked by cross-checking of paper files and the digital database. The database was imported and analysed by using the statistical software Stata SE14.The study was approved by the Health Department of the District Office of Rosenheim, where the study has taken place.The District Office has been acting as legal guardian of the involved unaccompanied refugee minors. In addition, the study has been granted ethical clearance by the Ethics Committee of the Ludwig-Maximilians-Universität, Munich (opinion number 462–16).n = 9/154) were female and 93.5% (n = 144/154) were male participants; for one person the sex was not recorded . The recorded age range was from 10 to 18 years at the moment of the first medical check-up, with a median age of 16 years . Figure This study included 154 URM, of which 5.8% (n = 72/154) of the children from locations A and B, whereas in location C the country of origin was not recorded. The URM came from 14 different countries. The main countries were Somalia with 27.8% (n = 20/72), Eritrea 20.8% (n = 15/72) and Afghanistan 19.4% (n = 14/72), followed by Syria 13.9% (n = 10/72)  and Eritrea (n = 4/9).The country of origin was documented for 46.7% n = 7/154 of tn = 40/48) of these URM had crossed the Mediterranean Sea in boats to reach Europe, the other 16.7% (n = 8/48) indicated to have fled through overland routes only.For 48 URM (31.2%) the routes of passage were documented. 83.3% (In 129 out of the 154 URM (83.7%) a clinical diagnosis statement was explicitly given by the examining physicians, Table In some participants more than one diagnosis was documented. Therefore the percentages add up to more than 100%. The suspicion of PTSD was raised in locations A and B only, however, since a structured assessment was only done in location A, only suspected cases from this location are listed. The category “Other” comprises : alopecia, asthma, cardiac murmur, conjunctivitis, ectoparasites , eczema, enterobiasis , eye trauma, gastroenteritis, hernia, hypertension, hypotension, jaundice, leishmaniasis, lymphadenitis, malaria, orchitis, scoliosis, skin ulceration, soft tissue abscess, thrombocytopenia.Not explicitly mentioned in Table n = 43/154) individuals the clinical diagnosis of scabies was recorded. In 9.1% (14/154) the individuals reported itchiness. All 4 referrals that were made to a dermatologist were due to scabies. Other findings included 2 cases out of 154 (1.3%) suspicious of cutaneous Leishmaniasis, one atopical eczema (0.6%) and one leg ulceration (0.6%).Altogether 49 of the 154 URM (31.8%) had a dermatological finding, in 27.9% (n = 30/42) of the children with a recorded dental status, were classified as needing an intervention through a dentist, with 3 of them being directly referred to a dentist.Almost three quarters  had no clinical findings. However, after considering all laboratory results, the TB diagnostics and the referrals to specialists only 12.3% (n = 19/154) had neither a recorded diagnosis, nor a pathological technical finding nor a recorded suspicious symptom.Within the cohort, 58 URM  the anti-HAV IgG only was determined, and in 75% of these (n = 12/16) the result was positive. In location B only in 2 URM the anti-HAV IgG was determined, in both cases with a negative result. In location C both the anti-HAV IgM and the anti-HAV IgG were measured. All anti-HAV IgM were negative and all anti-HAV IgG were positive in the whole sub-cohort (n = 65/65). Across all locations, 92.8% (n = 77/83) of the URM showed immunity against hepatitis A.In location A in 57.1% (n = 113/154) of the participants all three variables HBsAG, anti-HBs and anti-HBc were available. Among these, 8.0% (n = 9/113) were HBsAG positive and therefore considered to be chronic carriers of hepatitis B virus, although a recent infection and hence an acute HBV infection cannot be ruled out. In 92.0% (n = 104/113) HBsAg was negative. Only anti-HBs positive were 4.4% (n = 5/113), and 30.1% (n = 34/113) were anti-HBc positive regardless of anti-HBs status, and therefore considered to have been previously exposed to hepatitis B virus in the sense of a resolved infection. In 57.5% (n = 65/113) of the tested participants all three variables were negative and they were therefore considered naive with regard to exposure to hepatitis B virus. The following pie chart shows the HBV exposure status.In a total of 73.4% , a 16 year old URM had a positive result.In location C 100% (n = 65/65) of the UMR were tested for hepatitis C antibodies. One male  of the children the HIV status was determined and a syphilis screening through a TPHA test conducted. In all individuals the result for HIV was negative, while in 2.4% of the URM (n = 3/126) the TPHA test revealed a positive result; all 3 were male in the age range between 16 and 17 years.In 81.8%  of the individuals, with 25% (n = 7/28) stating to have had respective contacts. In addition, Quantiferon Gold Test was executed in 78.6% (n = 22/28) individuals, with one result (3.6%) returning positive in a URM who has not indicated prior Tb contact. No suspicion of tuberculosis appeared in the list of diagnoses here, no chest X-Rays were documented, and cough as an indicating symptom was not routinely documented in this location at all.In location A previous contacts to known Tb cases was documented in 82.1% (n = 6/61) of the URM were classified as suspect cases for Tb. Besides these suspect cases there were 8.2% (n = 5/61) of the URM with a documented positive Quantiferon test, which could be assumed to be latent infections.In location B the examining physician has been a specialist for pulmonology. Here both X-Ray and Quantiferon testing were conducted. In addition cough was documented. A total of 9.8% (n = 3/65) of the URM the suspicion of a case of tuberculosis was expressed and a referral to a specialist for pulmonology ordered.In location C there were neither Quantiferon test nor chest X-Ray conducted. However in 4.6% (n = 9/154) of the entire cohort suspected cases of tuberculosis were identified and 3.9% (n = 6/154) were supposedly latent infections.In 5.8% (n = 85/154) of the URM, while in 7.1% (n = 11/154) a parasitological finding was obtained. In 5.2% (n = 8/154) of the participants Gardia lamblia was identified, in 1.3% (n = 2/154) of the participants Schistosoma mansoni and in 0.6% (n = 1/154) of the participant Strongyloides stercoralis. In a total of 40.3% (n = 62/154) of the children an antigen test on Helicobacter pylori was performed in the stool sample, returning a positive result in 25.9% (n = 40/154).Routine stool diagnostics was conducted in 55.2% (n = 112/154) of the URM haemoglobin (Hb) was measured. In location A and B no anaemia was found, based on their threshold definition of 11 g/dl. In location C, 40.6% (n = 26/64) were classified as anaemic. However, this classification is based on higher threshold values as compared to locations A and B (see section “methods”). If the same threshold level as for locations A and B was to be applied, only 3 individuals would have been declared as anaemic. No pathological findings were recorded for the mean corpuscular volume (MCV).In 72.7% (n = 8/46) of the participants where differential blood count was performed. None of the individuals with intestinal parasites showed an eosinophilia.An increased eosinophil count was measured in 17.4% (n = 64/65) of the URM in location C only. The mean count was 6.61 × 103/μl (SD = 2.02). Only 3.1% (n = 2/65) of the URM had leukocyte counts above 10.0 × 103/μl. Their diagnoses were scabies and an ulceration of the leg.The level of the leukocyte count was measured in 98.5% (n = 64/65) of the URM the mean count was 288 × 103/μl .Thrombocyte counts as well were only available in location C. Here, in 98.5%  of the URM had no clinical findings. However, this subgroup of individuals diminished in numbers when all laboratory results, the TB diagnostics and referrals were included. Therefore a high overall morbidity of 87.7% with at least one clinical diagnosis or suspicious laboratory result was seen in this cohort, which is relatively high compared to data from Berlin where 56.6% had no pathological finding [About one third  of the children seemed to have been vaccinated in the past. Only in one  of the URM the level was high enough to assume long-term protection against hepatitis B [The status of immunity for hepatitis B was indicated through different serology constellations. In location C the exact anti-HBs levels were determined. This data allows the interpretation that only 3.2% (atitis B . The lown = 40/62) of the URM tested for Helicobacter pylori were positive. A previously conducted study described that mortality through e.g. stomach cancer is increased in migrant populations. Spallek et al. explained this risk through a widespread infection with Helicobacter pylori in children in developing countries [A total of 64.5%  in the cohort indicated to have undergone FGM. Especially with regard to the known countries of origin of all female URM of the cohort , this low prevalence is questionable. In a report from the Population Reference Bureau the prevalence of undergone FGM for females of the age group between 15 and 19 years was indicated for Somalia at 96.7% and for Eritrea at 78.3% . The medn = 7/28) of the URM from location A have suffered from a suspected post-traumatic stress disorder. Other studies analysed the prevalence of PTSD in children of refugees in Germany and showed that 19% had the full clinical picture of PTSD [The risk to develop PTSD is depending on the kind of trauma. About half of the victims of war, torture or flights are expected to develop PTSD, compared to about 10% of victims of traffic accidents, according to a guideline on PTSD from 2011 . In this of PTSD . Previou of PTSD . URM hav of PTSD , 37. Pre of PTSD .It has to be mentioned as a shortcoming of this study that PTSD itself was not diagnosed in this sub-cohort by a physician particularly trained in the detection of PTSD but by applying clinical judgement only, based on three key symptoms. The diagnosis should be more interpreted as an assessment of distress by addressing indicators comparable to a quick PTSD assessment. Nevertheless, these core symptoms are matching with the contents of commonly used questionnaires, such as the child PTSD symptom scale of the American Academy of Child and Adolescent Psychiatry . The focAn additional weakness of the study is the data pooling of all three sites for the data analysis, instead of the analysis location by location. It is very unlikely that the distribution of the URM to certain locations for their medical entry examination was geared by a substantial bias. It was assumed that the URM that entered Bavaria in the investigated time period represent a collective target population with comparable backgrounds.Certainly the disparities between the different assessment tools that were applied by the involved study sites and physicians may be summoned as a weakness of this study. However, the authors see this aspect rather as a finding of the study, and hope to contribute hereby to the future development of standardized tools to be employed in similar situations in the German health system when working with unaccompanied refugee minors as patients.This study shows the spectrum and extent of immediate health care needs of URM upon their arrival to Germany. The majority of the health conditions are common illnesses known to German physicians. At the same time the findings have to be interpreted in the light of the peak influx of refugees into the German state of Bavaria towards the end of 2015. Two key findings are the lacking of standardized algorithms and documentation for the medical coverage of URM in Germany, and considerable potential for primary and secondary prevention of illnesses through evidence based health interventions, such as vaccination against hepatitis B or psychosocial support in populations prone to suffering from PTSD.


Streptococcus species in the intestinal tract and a wider presence of Gram-positive cocci in jejunal biopsies. In particular, a higher number of Gram-positive cocci (of the genera Streptococcus and Staphylococcus), Eubacterium, Peptostreptococcus, and HSP60-producing Gram-negative microbes have been found in the stools of KS children, and their effects on the antigenic repertoire of specific T cells and Vβ2 T cell expansion have been assessed. Conversely, Lactobacilli were lacking in most children with KS compared with other febrile illnesses and healthy controls. All studies available to date have confirmed that an imbalance in the gut microbiota might indirectly interfere with the normal function of innate and adaptive immunity, and that variable microbiota interactions with environmental factors, mainly infectious agents, might selectively drive the development of KS in genetically susceptible children. Further investigations of the intestinal microflora in larger cohorts of KS patients will provide clues to disentangle the pathogenesis of this disease and probably indicate disease-modifying agents or more rational KS-specific therapies.Kawasaki syndrome (KS) is a necrotizing vasculitis of small- and medium-sized vessels mostly affecting children under 5 years of age; a host of clinical and epidemiological data supports the notion that KS might result from an infectious disease. However, many efforts have failed to identify a potentially universal trigger of KS. The contribution of the intestinal microbial community—called the “microbiota”—to KS has been evaluated by an increasing number of studies, though limited to small cohorts of patients. Differences in the microbiota composition were found in children with KS, both its acute and non-acute phase, with abnormal colonization by  The most insidious primary vasculitis in childhood is Kawasaki syndrome (KS), an acute multi-systemic illness which predominantly affects children under 5 years of age . CurrentKS incidence varies widely among different ethnic groups; for instance, in the United Kingdom it has stabilized and remains low at 2.8 per 100,000 population under the age of 20 years. However, general practitioners should be aware that the condition occurs throughout childhood and across the seasons, and—given the potential cardiovascular sequelae—KS should be considered in all children with persistent fever, even in older children and adolescents . There iThe main aim of this review was to analyze the relationship between the microbial community, or the “microbiota,” and the overall impact of bacterial or viral infections in the potential development of KS. Scientific papers have been searched from the electronic databases of PubMed until January 2019; the retrieving words were “Kawasaki disease,” “Kawasaki syndrome,” “microbiota,” and “microbiome”; additional reports were identified and analyzed through the specific references cited in the retrieved papers. Only papers published in English and those showing evidence-based data were included in our evaluation.The microbiota, a microbial community of trillions of microorganisms and at least 1,000 different bacterial species, some eukaryotic fungi and viruses, and which covers every surface of the human body, plays a contributory role in many infections, immune-mediated disorders, rheumatologic diseases, and disorders of the nervous system. The microbiome, on the other hand, is the collection of the whole genome sequences of those microorganisms, consisting of more than 5,000,000 genes , 15. In Enterococcus and Streptococcus, is then gradually replaced by anaerobes such as Bifidobacterium and Lactobacillus and finally reaches the adult composition dominated by Bacteroides and Firmicutes  and their related anti- and pro-inflammatory cytokines as well as several other mediators of inflammation or antimicrobial peptides, defensins, and secretory immunoglobulin A (IgA) . The intFaecalibacterium praunsitzii and increased numbers of Escherichia coli in IBD  are the most powerful T cell mitogens ever discovered, with potent immunostimulatory actions that simultaneously activate the major histocompatibility complex class II molecules and T cell receptors, leading to massive activation of various immune cells. Many studies have investigated the involvement of SAgs in KS. One of the first studies showed the selective expansion of T cell receptor (TCR) Vβ2-bearing T cells in the peripheral blood of children during the acute phase of KS . In addiStaphylococcus aureus and Streptococcus pyogenes in the pathogenesis of KS, finding numerous SAgs implicated, which brought the total number of the known staphylococcal SAgs to over 20 and streptococcal SAgs to 12 , such as SPE-A. They showed that KS patients had significant elevation of IgM antibodies against one or more of five SAgs throughout the first to the fourth disease week . In a fugs to 12 .Streptococcus pyogenes in the stools of patients with KS. Stool specimens were obtained from 36 patients with KS during the acute phase and 26 age-matched healthy children. The authors examined genes related to five Sags—SPE-A, SPE-C, SPE-G, SPE-J, and TSST-1—using polymerase chain reaction; throat and stool cultures were assessed to evaluate the presence of Streptococcus pyogenes and Staphylococcus aureus in KS patients. They reported that at least one of the SAg-related genes was detected more frequently in the stools of children with KS (Mycoplasma pneumoniae antibody (>1:640), suggesting a potential role of Mycoplasma pneumoniae in KS and the importance of anti-Mycoplasma treatment , induce a coronary arteritis in mice similar to that observed in KS, Sato et al. evaluated the role of a free-β-glucan diet on CAWS-induced vasculitis and found that quality of diet might affect the progression of systemic vasculitis  and 2 kinds of staphylococci (Staphylococcus capitis and Staphylococcus hyicus) were isolated only from KS patients, suggesting that some antigens inducing a delayed-type hypersensitivity reaction in the mucosa might inundate the body by breaching the barrier of the small intestinal mucosa of KS patients  was significantly lower (p < 0.001) than in the other cohorts. Moreover, no significant differences in the presence of Staphylococcus, Streptococcus, Enterococcus, Enterobacteriaceae, Bifidobacterium, Clostridium, Veillonella, or Bacteroides were found among the three groups. Conversely, the presence of Eubacterium and Peptostreptococcus was significantly higher in KS patients than in patients with other febrile diseases , though no significant differences were observed between KS patients and healthy children (Lactobacillus species are anti-inflammatory and beneficial for health, particularly during the first years of life, due to their action in protecting against colitis, reducing pro-inflammatory cytokines such as TNF-α, IL-1, or IL-6, and increasing the subsets of regulatory T cells (Lactobacilli in maintaining the epithelial homeostasis of the gut and their striking anti-inflammatory potential (Due to the fact that the gastrointestinal tract is the largest interface between microbial factors and their host, containing the largest proportion of bacteria and the largest amount of lymphoid tissue in the body, it was hypothesized by Eladawy et al. that the intestinal milieu could be altered in children with KS, and indeed KS patients have a higher incidence of gastrointestinal symptoms and complications . Specifichildren . This ob T cells . In factotential , 92.in vitro (Nagata et al.  also stuin vitro . This miRuminococcus, Roseburia, and Faecalibacterium were mostly predominant during the non-acute phase, while a higher presence of Streptococcus spp., including Streptococcus pneumoniae, pseudopneumoniae, mitis, oralis, gordonii, and sanguinis, was detected in the fecal samples during the acute phase (In order to evaluate the differential microbiota composition of KS patients, Kinumaki et al.  performete phase .Streptococcus spp. have been demonstrated (Streptococcus spp., and that changes in the gut microflora composition might promote systemic and extra-intestinal inflammation. Therefore, all presented studies confirm the concept that an imbalance in the gut microbiota might directly or indirectly interfere with the normal functions of the immune system, and that the interaction with other environmental factors, mainly infectious agents, might lead to the final development of KS.This novel interpretation of a disease can be shared by other conditions, as liver cirrhosis and Sjögren syndrome, in which major changes of gut microbiota with higher proportions of nstrated , 96. TheAs no etiologic agent has ever been accused of being directly involved in the etiology of KS, basic research evaluating the pathogenic mechanisms of this disease will probably provide better therapies and probably consent to identify the most vulnerable hosts and protect them. A potential relationship between eubiosis and the protean functions of immunity has been contemplated by different studies, and conversely a relationship should exist between dysbiosis and immunity dysfunction shown by various diseases . It is sSE, IP, and DR contributed to all stages of the preparation of this manuscript, including conception and writing. All authors approved the final submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.