Soil erosion can affect the distribution of soil nutrients, which restricts soil productivity. However, it is still a challenge to understand the response of soil nutrients to erosion under different soil types.2O3, CaO, Fe2O3, K2O, Na2O, MgO, TiO2, and SiO2), were analyzed in the profiles from yellow soils, red soils, and lateritic red soils in an erosion region of Southeast China. Soil erodibility K factor calculated on the Erosion Productivity Impact Calculator (EPIC) model was used to indicate erosion risk of surface soils (0∼30 cm depth). The relationships between these soil properties were explored by Spearman’s rank correlation analysis, further to determine the factors that affected the distribution of SOC, SON, and soil major elements under different soil types.The distribution of soil nutrients, including soil organic carbon (SOC), soil organic nitrogen (SON), and soil major elements (expressed as Al2O3 and SiO2, in the yellow soils, were significantly larger than those in the red soils and lateritic red soils. Moreover, the concentrations of major metal elements positively correlated with silt proportions and SiO2 concentrations positively correlated with sand proportions at the 0∼80 cm depth in the yellow soils. Soil major elements depended on both soil evolution and soil erosion in the surface layer of yellow soils. In the yellow soils below the 80 cm depth, soil pH positively correlated with K2O, Na2O, and CaO concentrations, while negatively correlated with Fe2O3 concentrations, which was controlled by the processes of soil evolution. The concentrations of soil major elements did not significantly correlate with soil pH or particle distribution in the red soils and lateritic red soils, likely associated with intricate factors.The K factors in the red soils were significantly lower than those in the yellow soils and significantly higher than those in the lateritic red soils. The SON concentrations in the deep layer of the yellow soils were twice larger than those in the red soils and lateritic red soils, while the SOC concentrations between them were not significantly different. The concentrations of most major elements, except AlThese results suggest that soil nutrients and soil erodibility K factor in the yellow soils were higher than those in the lateritic red soils and red soils. The distribution of soil nutrients is controlled by soil erosion and soil evolution in the erosion region of Southeast China. We hypothesized that both soil erosion and soil evolution control the distribution of soil nutrients in the erosion region of Southeast China. This study is closely associated with the conservation of soil nutrients, which is critical for ecological stability . Therefo2  is located in Fujian Province, with a drainage area of 14,741 km2 . The ave2 . The ori2 . Over 902 . These s2 .Soil samples were collected in the winter season of January 2018. The three soil sites were selected according to different soil types, including red soils, lateritic red soils, and yellow soils, respectively . The soi4, and three mL HNO3 at 120 °C for 3 days  in the soils  were removed by treating with 0.5 mol Lfor 24 h , and inofor 24 h . The tret acre h/100 acre/ft/tanf/in. For brevity, the unit of the K factor will not be noted.Soil erodibility can be used to indicate the erosion risk of surface soils because of the significant quantitative relationship between the amount of soil loss and the K factor . Many mo2O3, CaO, Fe2O3, K2O, Na2O, MgO, TiO2, and SiO2), and the K factor values in the red soils, lateritic red soils, and yellow soils. One-way ANOVA with the least significant difference (LSD) test was performed to determine the differences in the proportions of different-sized particles, soil pH values, the concentrations of SOC, SON, and soil major elements, and the K factor values among the three soil types at the level of P < 0.05. All data were calculated via the Kolmogorov–Smirnov test, which is one of the non-parametric tests commonly applied to analyze the normal distribution of the sample data set  in the upper layer. It is can be inferred that the re-assimilation of 15N-depleted inorganic N through microbial immobilization into microbial biomass  than those in the red soils and lateritic red soils . The larN values . The C/NN values , but it N values . The C/N biomass , resultiep soils .+, K+, Ca2+, and Mg2+ are preferentially dissolved and lost under strong leaching conditions, while the iron and aluminum, these have a low mobilization, are gradually accumulated with soil evolution . In the deep soils (below 80 cm depth), the SON concentrations in the yellow soils were twice higher than those in the red soils and lateritic red soils, which was mainly attributed to the re-assimilation of inorganic N through microbial immobilization. The concentrations of soil major elements in the three soil types were almost lower than the background values of major elements in the mean soils of Fujian Province. Moreover, the concentrations of most major elements in the yellows were significantly larger than those in the red soils and lateritic red soils. The variation of soil major elements in the surface layer of the yellow soils was affected by both soil erosion and soil evolution, while it in the deep soils was mainly affected by soil evolution. These results suggest that yellow soils have a higher level in soil nutrients and soil erodibility compared to the red soils and lateritic red soils under the erosion region of Southeast China.10.7717/peerj.11630/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11630/supp-2Supplemental Information 2t acre h/100 acre/ft/tanf/in. YS, yellow soil; LRS, lateritic red soil; RS, red soil.The unit of the K factor is Click here for additional data file.


Mycobacterium ulcerans, is a devastating necrotizing skin disease. Key to its pathogenesis is mycolactone, the exotoxin virulence factor that is both immunosuppressive and cytotoxic. The discovery that the essential Sec61 translocon is the major cellular target of mycolactone explains much of the disease pathology, including the immune blockade. Sec61 inhibition leads to a loss in production of nearly all cytokines from monocytes, macrophages, dendritic cells and T cells, as well as antigen presentation pathway proteins and costimulatory molecules. However, there has long been evidence that the immune system is not completely incapable of responding to M. ulcerans infection. In particular, IL-1β was recently shown to be present in BU lesions, and to be induced from M. ulcerans-exposed macrophages in a mycolactone-dependent manner. This has important implications for our understanding of BU, showing that mycolactone can act as the “second signal” for IL-1β production without inhibiting the pathways of unconventional secretion it uses for cellular release. In this Perspective article, we validate and discuss this recent advance, which is entirely in-line with our understanding of mycolactone’s inhibition of the Sec61 translocon. However, we also show that the IL-1 receptor, which uses the conventional secretory pathway, is sensitive to mycolactone blockade at Sec61. Hence, a more complete understanding of the mechanisms regulating IL-1β function in skin tissue, including the transient intra-macrophage stage of M. ulcerans infection, is urgently needed to uncover the double-edged sword of IL-1β in BU pathogenesis, treatment and wound healing.Buruli ulcer (BU), caused by  Mycobacterium ulcerans. It typically presents as painless, ulcerative skin lesions or as pre-ulcerative nodules, plaques and in oedematous forms (Buruli ulcer (BU) is a neglected tropical disease resulting from subcutaneous infection by us forms . This enus forms , 3. The us forms . Untreatus forms . More seus forms .M. ulcerans  by the Sec61 translocon  Figure, 38, 39,M. ulcerans infections , and iNOS, the inducible nitric oxide synthase that generates bactericidal NO in macrophages, both increase susceptibility to infection. Interestingly, SNPs in three genes involved in the autophagy pathway also affect BU. Two polymorphisms have been identified in the E3 ligase PARK2 which increase susceptibility to BU while one in NOD2 increases the risk of severe disease   . This lad lipids .M. ulcerans, or mycolactone-containing EV exposure, it has been demonstrated that priming of macrophages is probably via activation of TLR2, since genetic knockout of this receptor ablated the response  (We now confirm that mycolactone induces IL-1β release by macrophages  mycolacttis (RA) , 71–76, tis (RA) ]. ConseqOnce produced, several mechanisms for secretion of IL-1β have been proposed. All of these are independent of the canonical secretory pathway and Sec61-dependent translocation of proteins into the ER, and are known as unconventional secretory pathways  Figure. Recent Hence, the recent discovery of IL-1β in BU lesions, and its induction by mycolactone in macrophages is entirely in line with our current knowledge of the cell biology and biochemistry of Sec61 inhibition by mycolactone.M. ulcerans infection, by enhancing the activation state of surrounding cells, including innate immune macrophages and neutrophils. In the mouse footpad model of M. ulcerans infection, treatment with the non-specific steroid anti-inflammatory dexamethasone reduced footpad swelling  and down-regulating anticoagulant proteins such as thrombomodulin and the EPCR. IL-1β also induces procoagulant PAI-1, enhances tissue factor expression, and promotes angiogenesis via upregulation of adhesion molecule and vascular endothelial growth factor (VEGF) expression . This work was also supported by a grant from the Kennedy Institute Trustees.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.


CD40 is a costimulatory molecule that is key for the activation of antigen-presenting cells and other innate immune cells. It plays an important role in anti-tumor immunity, and agonists of CD40 have been shown to eliminate tumors in both pre-clinical and clinical settings, alone and in combination with other treatment modalities. Here we assess the expression of CD40 and associations with other mediators of immunity in a variety of tumor types and review the potential of CD40 agonists for cancer treatment, given the promise of enhancing the interplay between innate and adaptive immunity.CD40 is expressed on a variety of antigen-presenting cells. Stimulation of CD40 results in inflammation by upregulation of other costimulatory molecules, increased antigen presentation, maturation (licensing) of dendritic cells, and activation of CD8+ T cells. Here we analyzed gene expression data from The Cancer Genome Atlas in melanoma, renal cell carcinoma, and pancreatic adenocarcinoma and found correlations between CD40 and several genes involved in antigen presentation and T cell function, supporting further exploration of CD40 agonists to treat cancer. Agonist CD40 antibodies have induced anti-tumor effects in several tumor models and the effect has been more pronounced when used in combination with other treatments . The reduction in tumor growth and ability to reprogram the tumor microenvironment in preclinical models lays the foundation for clinical development of agonistic CD40 antibodies  that are currently being evaluated in early phase clinical trials. In this article, we focus on CD40 expression and immunity in cancer, agonistic human CD40 antibodies, and their pre-clinical and clinical development. With the broad pro-inflammatory effects of CD40 and its ligand on dendritic cells and macrophages, and downstream B and T cell activation, agonists of this pathway may enhance the anti-tumor activity of other systemic therapies. In the past decade, oncologic care has changed dramatically as immunotherapies have been developed for multiple tumor types. Immune checkpoint inhibitors targeting the programmed cell death protein 1(PD-1)/programmed cell death ligand 1 (PD-L1) pathway have significantly increased survival in randomized trials. Although durable responses are observed, most patients are inherently resistant or develop resistance to checkpoint inhibition over time . CombinaCD40 is constitutively expressed as a transmembrane receptor on DCs, myeloid cells, and B cells, as well as several types of non-hematopoietic cells, such as endothelial cells, fibroblasts, epithelial cells, and certain types of malignant cells ,17. The Engagement of CD40 by CD40L promotes the necessary clustering of CD40 receptors on the cell surface that initiates downstream signaling by one of two mechanisms: (a) activated CD40 binds Janus kinase 3 (JAK3) that in turn activates signal transducer and activator of transcription 5 (STAT5)-mediated transcription or (b) activated CD40 engages TNF receptor-associated factors (TRAFs) that can initiate nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways in immune cells. The specific downstream signaling resulting from CD40 activation is cell-type dependent ,15,17,21JAK-STAT pathway signaling is involved in cytokine-mediated signal transduction. CD40-CD40L binding on monocytes recruits JAK3 that phosphorylates and activates STAT5 to initiate transcription of target genes . JAK3-STInduction of NF-κB signaling pathway by binding of CD40 to CD40L induces activation of NF-κB inducing kinase (NIK). NIK activates IκB kinase α (IKKα), and IKKa in turn phosphorylates p100 to produce p52. P52 associates with relB and translocate to the nucleus to induce gene expression . NF-κB sMAPK signaling plays a role in the initiation of innate immunity in various cells to activate adaptive immunity (. p38) MASurvival of DC is mainly mediated by the PI3K pathway . On monoThe downstream signaling effects of CD40/CD4L are therefore diverse and differ between cell types.CD40 activation on DCs and monocytes upregulates the expression of other costimulatory molecules such as CD80 and CD86. Their effects on MHC molecules enhance antigen presentation, license DCs, and activate CD8+ T cells ,34,35,36CD40/CD40L binding, therefore, activates multiple innate immune cells, including DCs, macrophages, NK cells, and B cells, resulting in CD8+ and CD4+ T cell stimulation. Activation of cytotoxic and effector T cells leads to cell kill and apoptosis. n = 20,501) in samples from 534 clear cell renal cell carcinomas (ccRCC), 456 cutaneous melanomas, and 178 pancreatic adenocarcinomas, selecting tumor types that tend to be responsive to immunotherapy (melanoma and ccRCC) versus pancreatic adenocarcinoma which is resistant. Applying a cutoff Spearman correlation rho of ≥ 0.3 and an adjusted p-value of <0.001, we found that 198 genes in ccRCC, 361 genes in melanoma, and 935 genes in pancreatic adenocarcinoma were correlated with CD40 gene expression. The 50 genes with the highest correlation to CD40 in each cancer type are listed in The important role that CD40 signaling plays in anti-cancer immunity prompted us to explore genes that may be included as potential co-targets with a CD40 agonist-based therapy. We used RNA sequencing data from The Cancer Genome Atlas (TCGA) database to analyze the correlation of CD40 expression and that of other protein-coding genes  is a receptor for the cytokine IL-27. Although the role of IL-27RA in cancer is unknown, preclinical studies have shown that IL-27 inhibits tumor cell growth ,46. Tripn = 82) and include several C-X-C chemokine receptors including CXCR3, which is the ligand for CXCL10, as well as CXCR4 and CXCR6. We found several genes that are involved in the cytotoxic activity of T cells and NK cells including granzymes GZMM, GZMA, GZMH, and natural killer cell granule protein 7 (NKG7), that were similarly correlated with CD40 expression. NKG7 is a cytolytic-related protein expressed in NK cells and T cells, preferentially those polarized to Th2 direction ..106].Studies of the various CD40 agonistic antibodies remain in the early phase, and randomized data are not available yet. The activity has been seen as monotherapy, or in combination with other drugs, but optimal combinations and disease settings have yet to be established.Pharmacological activation of CD40 by antibodies activates signaling cascades in a variety of immune cells, particularly dendritic cells, B cells, and macrophages, resulting in increased inflammation and activation of cytotoxic T cells. Co-stimulation of innate and adaptive immunity might result in enhanced anti-tumor activity, and CD40 agonistic antibodies have therefore been the subject of intense preclinical and clinical research. Pre-clinical models assessing CD40 agonists alone or in combination with radiation, chemotherapy, immune-therapy, or angiogenic therapy have largely demonstrated superior activity to either modality alone. Additional pre-clinical studies are needed to further determine the mechanisms of action of these drug combinations and refine the dosing and sequencing of drugs and regimens. Early phase clinical trials of CD40 agonists in a variety of tumor settings have demonstrated activity, and additional clinical studies are needed to determine whether CD40 agonists can be added to the expanding repertoire of cancer drugs to either increase response (and preferably cure rates) in the frontline setting or to treat patients whose tumors have progressed on other frontline regimens.The importance of signaling of CD40/CD40L has been demonstrated in a variety of tumor models, and activation of CD40 affects multiple innate immune cells, providing the promise of harnessing CD40 as a therapeutic target in cancer. Early phase clinical trials have demonstrated that CD40 agonistic antibodies are generally safe, when administered alone and in combination with other anti-neoplastic modalities. Further studies are warranted to extensively evaluate CD40 agonists in cancer patients.


Nanoparticles generated during laser material processing are often seen as annoying side products, yet they might find useful application upon proper collection. We present a parametric study to identify the dominant factors in nanoparticle removal and collection with the goal of establishing an in situ removal method during femtosecond laser machining. Several target materials of different electrical resistivity, such as Cu, Ti, and Si were laser machined at a relatively high laser fluence. Machining was performed under three different charge conditions, i.e., machining without an externally applied charge ) was compared to machining with a floating potential and with an applied field. Thereby, we investigated the influence of three different charge conditions on the behavior of laser-generated nanoparticles, in particular considering plume deflection, nanoparticle accumulation on a collector plate and their redeposition onto the target. We found that both strategies, machining under a floating potential or under an applied field, were effective for collecting laser-generated nanoparticles. The applied field condition led to the strongest confinement of the nanoparticle plume and tightest resulting nanoparticle collection pattern. Raster-scanning direction was found to influence the nanoparticle collection pattern and ablation depth. However, the laser-processed target surface remained unaffected by the chosen nanoparticle collection strategy. We conclude that machining under a floating potential or an applied field is a promising setup for removing and collecting nanoparticles during the machining process, and thus provides an outlook to circular waste-free laser process design. Over the past decade, ultrafast laser ablation has emerged as a simple method for the direct generation of nanoparticles. The mechanism of femtosecond laser-assisted material removal is complex and has already been explored in detail ,2,3. ManThe effectiveness of copper as a broad antimicrobial  agent has been known since ancient times . RecentlNow, considering that nanoparticles are an essential resource for the above-mentioned applications but constitute a poorly managed waste stream in laser surface machining, the paradigm of a circular economy comes to mind . This leZheng et al., introduced a new technique to reduce the amount of Si nanoparticle redeposition onto a femtosecond laser machined target by using an external electric field . In thisIn this report, we build upon Zheng et al.’s study and investigate nanoparticle collection and removal efficiency during femtosecond laser machining by applying either a high strength electric field or a varying electric field between the collection plate and target. While the collection of Cu nanoparticles is of primary interest, we compare two more target materials of different electrical resistivity. The effect of the polarity of the applied electric charge and the laser scanning direction on nanoparticle collection efficiency is also considered. The comparison and assessments are carried out on the basis of photography of the collection plate upon machining, high-speed videography of the nanoparticle plume deflection, scanning electron microscopy (SEM) and confocal microscopy of the resulting target surface topography.3 Ω·m). Unpolished copper collection plates  with dimensions of 50 mm × 10 mm were used to collect the generated nanoparticles. All the target samples and the collection metals were ultrasonically cleaned in acetone for 5 min before laser experimentation. The scanning electron microscope images of each of the target material’s surface and the collection plate surface are shown in Two metals and a semiconductor target were used in this study to generate laser-ablated nanoparticles: copper , titanium  and silicon . The target materials were selected according to their electrical resistivity ranging from low  to intermediate  and to high  of 1 kHz. The laser energy per pulse (E) was reduced to a desired lasing energy using a computer-controlled attenuator composed of a polarizing beam splitter and half-wave plate. The horizontally polarized Gaussian beam (TEM00) was then focused down to 40 µm spot diameter (thF) of the respective target materials which were raster scanned under the stationary beam. The ablation threshold of each material was experimentally determined by plotting the square of the laser-irradiated line width as a function of the logarithm of the incident laser fluence [2, respectively.Samples were machined in air using an amplified Ti: Sapphire solid-state laser system  operating at a central wavelength of 800 nm, a pulse duration of ~100 fs and a repetition rate , which is schematically represented in The nanoparticle plume was filmed using a high-speed camera  equipped with an 18–108 mm macro zoom lens . The camera was temporally synchronized with the femtosecond laser system. The videos were gamma corrected, the region of interest was fixed, and each frame was converted into a .tiff image using PFV software . These processed images were then fed into a MATLAB code. An intensity threshold was set to distinguish between bright (plume) and dark (background) pixels. Upon identification of the bright pixels and their center points, the plume deflection angle of each frame was determined by fitting the center of bright pixels into a first order polynomial function.The machined surfaces were imaged with a scanning electron microscope (SEM)  both immediately after laser micromachining and once again after having been ultrasonically cleaned in acetone for 10 min. 3D confocal microscopy  was carried out to determine the ablation depth on the target. The depth of all the machined surfaces relative to the pristine surface were measured using the LEXT software , as shown in All the results presented in the following stem from experiments carried out with parallel laser scanning direction. From Finally, we note that collection seems to be directly correlated with the materials’ electrical properties , which are a signature of the number of free electrons present on the material: the seemingly densest nanoparticle packing and maximum collection spread were observed for copper, which has the lowest electrical resistivity among the studied materials, whereas the smallest collection spread with seemingly looser packing was exhibited by Si, which has the highest electrical resistivity. We suggest that the appearance of the nanoparticle packing density and collection spread provide an indication of the absolute number of nanoparticles collected. A study by Irimiciuc et al., showed that an increase in electrical conductivity/decrease in resistivity on metals led to a release of more charge volume and a more intense electrostatic field, which again results in a greater ablation . The higWe acknowledge that photographs merely provide a 2D presentation of a 3D phenomenon, and thus only allow for qualitative comparisons. Ideally, quantitative measures substantiated these conclusions drawn from the “look” of the photographs. Unfortunately, our current experimental setup does not include a sufficiently precise force sensor to make significant in situ high-precision weight measurements of the nanoparticle agglomerates, and any attempts to carefully transfer collection plates from their holder to another instrument to conclusively assess and compare the collected nanoparticle weight proofed ineffective and error-prone. Furthermore, in situ microscopic imaging methods with possible 3D reconstruction would enable a direct analysis of the spatial distribution of the collected nanoparticles.Next, we studied the dependence of nanoparticle collection on the electric field intensity. The nanoparticles plume deflection was visualized using a high-speed camera synchronized with the femtosecond laser system.Conversely, a confinement in the nanoparticle plume was observed in the presence of a floating potential b and appWhen considering the electric field condition, the potential difference across the target and collection plates is definite at ±2000 V. Under such conditions, the electric charge accumulated during the laser ablation process is constant from pulse to pulse, resulting in a steady local electric field which controls the trajectory of the ionized species: the nanoparticles generated during the process of laser ablation. Such a strong and steady local electric field explains the high degree of confinement and the weak angular deflection of the nanoparticle plume c seen unIn contrast, neither the conventional PLD condition nor the floating potential conditions are electrically well defined, since the potential difference across the target and collection plates varies from pulse to pulse. The induced electric field will completely depend on the amount of charge left over from the previous pulse (effective field). Thus, this varying local field from pulse to pulse will cause diverse effects on the ionized particle; the behavior of the resulting nanoparticle plume will accordingly be correlated with the varying local electric field intensity. The local electric field for the conventional PLD machining conditions is expected to be weaker than the electric field resulting from the floating potential with an externally applied voltage of ±2000 V because of a relatively small charge accumulation during the laser ablation process. Thus, the differences in local field intensity of an externally applied floating potential compared to that observed under conventional PLD conditions explain the greater nanoparticle plume confinement and the weaker variation in the deflection of the nanoparticle plume. The electric charge accumulated during the laser ablation process is seemingly small; hence, the local electric field induced during the conventional PLD condition is less intense in comparison with the floating potential condition counterpart.That the applied field intensity influences the nanoparticle plume was further confirmed through plume videography, as seen in A confinement in the nanoparticle collection pattern is also observed under increasing floating potential c. The coHaving investigated the nanoparticle collection patterns on the collector plates, we now present the effects of the machining conditions on the appearance of the laser-machined target surfaces.Directly after laser machining, i.e., before ultrasonication, we observe that the actual topography of the machined surface shows some variation with the actual machining conditions a–c. The While the experimental conditions under investigation here showed no impact of ultrasonication on the laser-induced surface structure after removal of the remaining nanoparticles by, the ablation depth—as determined by confocal microscopy—indicated considerable effects due to the actual experimental conditions. Detailed experimental results are presented in the The laser machining experiments were performed with two different laser scanning directions  as depicted in Conversely, for the perpendicular case, the distance between the nanoparticle generation sites along the long scan lines and the short edge of the charged collection plate varies. As a consequence, the force acting on the nanoparticles due to the charged collection plate varies throughout the machining of a single long scan line. When the laser incidence position on the target moves away from the short edge of the collector plate, the force decreases; when it approaches the collector plate again in the return scan, the force successively increases. As a result, initially ablated nanoparticles are attracted to a single spot on the collector plate, which is where the initial agglomeration starts and grows into a significant protrusion. The nanoparticles produced during later laterally translated scans will then drive towards this initially generated agglomerate protrusion rather than to the pristine collector plate which is at a slightly farther distance. As a consequence, the perpendicular scanning protocol results in a narrower collection pattern on the Cu collection plate.Plume videography showed no significant differences between the two scanning protocols compare .The comparison of SEM micrographs of the surfaces machined with parallel and perpendicular scanning direction for a given material after sonication revealed that the scanning direction does not affect the laser-induced structures compare . Yet, whIn this study, we carried out a parametric study to identify the dominant factors in nanoparticle collection and removal with the goal of establishing therefrom an in situ removal method during femtosecond laser machining. The influence of actual laser machining conditions  in removing the laser-produced nanoparticles, collecting them on a Cu collector, and the respective nanoparticle plume deflection behaviors were investigated. The connection between the different surface properties of the machined surface (topography before and after ultrasonication and the ablation depth) and the actual machining conditions were also explored.Machining in the presence of a floating potential results in a broader nanoparticle collection pattern for all tested targets  in contrast to machining in the presence of an applied field, where nanoparticle collection appears confined to a very small area on the collection plate. This observation correlates with a stronger confinement and a weaker angular deflection in the nanoparticle plume that was observed in proximity of an applied field in comparison with the floating potential condition. Under the conventional PLD condition in air, the nanoparticle plume was found to expand freely. The strong and steady local electric field induced under the applied field condition in contrast to the varying local field under the floating potential condition explains the high degree of confinement of the nanoparticle plume and the weak angular deflection of the nanoparticle plume. Furthermore, an increasing confinement of the nanoparticle plume and the resulting nanoparticle collection pattern was also observed with an increase in the applied field intensity and floating potential. Qualitatively judged from the collector plate photographs, the nanoparticle collection quantity directly correlates with the materials’ electrical properties . Furthermore, the actual accumulation pattern on the Cu collector varies from one material to another, which is particularly evident for the collection patterns obtained upon machining with a floating potential. The topography of the machined surface was examined using SEM and the ablated depth was analyzed with the aid of a confocal microscope. Our results confirm that the surface topography of the machined surface before sonication and the ablation depth strongly depend on the actual charge conditions during machining. Yet, from our experiments, we found no notable impact of the charge condition during laser machining on the resulting laser induced surface textures, which were previously hidden under agglomerated and un-sintered nanoparticles. Whether an electric field or a floating potential is better suited to collect higher amounts of otherwise volatile nanoparticles will be an interesting subject of future studies.Such experimental efforts might benefit from a setup inside a closed system with a larger collection plate. Ideally, both target and collector are connected to high-precision force sensors. In addition, extremely valuable information would be gained if an alike experimental setup also allows for in situ imaging and sizing of the redeposited and collected nanoparticle clusters.Based on our findings, we conclude that machining metals and semi-conductors, in particular, under the floating potential or applied field condition with a parallel laser raster scanning protocol is a promising route for removing and collecting nanoparticles during the machining process. The current technique designed for collecting a substantial amount of such laser-generated nanoparticles in air directly during surface machining and patterning without affecting the target surface provides an outlook to circular waste-free process design.


The aim of the present study was to analyze the prevalence, epidemiology and relevance of shoulder injuries in polytraumatized patients in a large national trauma database.We hypothesize a high prevalence of shoulder injuries in traffic accidents and a high prevalence of concomitant injuries of the thorax leading to an aggravated clinical course and higher Injury Severity Score (ISS). Furthermore, we hypothesize an increased rate of surgical treatment with the severity of the injury.The retrospective analysis is based on the database (2002–2013) of the TraumaRegister DGU® and includes statistical data from 608 hospitals. The severity of injuries and trauma were scaled using the Abbreviated Injury Scale (AIS), and the Injury Severity Score (ISS), respectively. Patients with an ISS ≥ 16 were included in the study, and injuries were subdivided according to their anatomical involvement and analyzed with respect to the trauma mechanism and the resulting injuries.In this study, 54,076 cases of patients with an ISS ≥ 16 were analyzed. Shoulder injuries occurred in 15,115 patients (27.9%). Of these, 68.5% were caused by traffic accidents, especially in motorbike, bicycle, and pedestrian accidents. We found more shoulder injuries in blunt trauma mechanisms. Moreover, patients with shoulder injuries spent on average 1.7 more days on the intensive care unit (ICU), or intermediate care unit (IMCU), according to the severity of the injury, and had longer overall hospital stays (26.2 vs. 24.1 days) than patients without shoulder injuries. The overall ISS was increased in patients with shoulder injuries, whereas an increase of mortality could not be identified. Concomitant thoracic injuries occurred significantly more often in patients with shoulder injuries (82.9% vs. 69.6%). Injuries of the abdomen, pelvis, and lower extremity showed no correlation with shoulder injuries, whereas head and spine injuries showed a significant correlation.Shoulder injuries are very common in polytraumatized patients. Together with their distinctive concomitant injuries, they have an aggravating impact on the clinical progress. Our data confirm the correlation with thoracic injuries. Furthermore, we identified an increased risk of shoulder injuries in motorbike, bicycle, and pedestrian accidents. An increase in mortality could not be identified. Severe trauma is the sixth leading cause of death worldwide, and among young adults under 35 years of age, it is the leading cause of death and disability . This chIn major trauma, injuries to the shoulder, including severe nerve and vessel injuries, are frequently seen  and may In the present literature, several studies highlight the relevance of scapular fractures in a severe trauma or shoulder injuries in general –15. NeveThe purpose of this study was to investigate the prevalence and clinical features of shoulder injuries in polytraumatized patients. Furthermore, the impact on mortality was assessed, and concomitant injuries were identified.We presumed that, in the treatment of severely injured patients, the early identification of patients with a high risk for shoulder injuries might help reduce delayed diagnoses and decrease complications. We hypothesized a high prevalence of shoulder injuries in traffic accidents and a high prevalence of concomitant injuries of the thorax leading to an aggravated clinical course and higher ISS.The TraumaRegister DGU® (TR-DGU) of the German Trauma Society [Deutsche Gesellschaft für Unfallchirurgie (DGU)] was initiated in 1993. The aim of this multi-center database is to provide a pseudonymized and standardized documentation of severely injured patients. Data are collected prospectively in four consecutive time phases from the site of the accident until discharge from hospital: (a) Pre-hospital phase, (b) emergency room and initial surgery, (c) intensive care unit, and (d) discharge. The documentation includes detailed information on demographics, injury pattern, mechanism of injury, comorbidities, pre- and in-hospital management, a course on intensive care unit, relevant laboratory findings including data on transfusion, length of hospital stay, and outcome of each individual. The inclusion criterion is admission to hospital via an emergency room with subsequent ICU/IMCU care or reaching the hospital with vital signs but dying prior to admission to ICU/IMCU.The infrastructure for documentation, data management, and data analysis is provided by the Academy for Trauma Surgery , an affiliation of the German Trauma Society. The scientific leadership is provided by the Committee on Emergency Medicine, Intensive Care and Trauma Management (Section NIS) of the German Trauma Society. The participating hospitals submitted their data pseudonymized into a central database via a web-based application. Scientific data analysis is approved according to a peer-review procedure established by Section NIS.The participating hospitals are primarily located in Germany (90%), but an increasing number of hospitals from other countries contribute data as well. Currently, more than 33,000 cases from over 600 hospitals are entered into the database each year.Participation in TraumaRegister DGU® is voluntary. For hospitals associated with the trauma network of the DGU®, however, the entry of at least a basic data set is obligatory for reasons of quality assurance.The present study is in line with the publication guidelines of the TraumaRegister DGU® and was registered as TR-DGU project ID 2014–029.In Germany, 608 participating hospitals contributed their data to the TR-DGU in the dataset from 2002–2013. All patients with an initial ISS ≥ 16 that were documented in the TR-DGU from 2002–2013 were included in the present study. Excluded were all patients with an ISS ≥ 16 due to a single craniocerebral injury (CCI). In sum, 54,076 patients were included in the study. Patients were divided into two subgroups: the “shoulder” group with patients presenting shoulder injuries , and the “non-shoulder” group which included patients without the presence of any shoulder injuries . The severity of injuries was documented and coded according to the 2005 revised version of the AIS [Concerning the outcome and specification of injury, the average Revised Injury Severity Classification II (RISC II), mean ISS, mortality, trauma mechanisms, and further clinical and epidemiological data  transfusion, Glasgow Come Scale (GCS), and hospital stay including intensive care unit (ICU) and intermediate care unit (IMCU) were assessed. The concomitant injury pattern of polytraumatized patients demonstrates the prevalence of concomitant injuries (any injury of the specified anatomic region with an AIS score ≥ 2) that occurred in conjunction with a shoulder injury.t test for continuous data, whereas the Pearsons’ Chi-Square test was used for categorical data. Continuous values are presented as mean ± standard deviation (SD).Differences between the groups were evaluated with Student’s p value ≤ 0.05 (two tailed) was considered to be statistically significant. The statistical analyses were performed with SPSS .A n = 15,115, 27.9%). Patients in the shoulder group had a significantly increased ISS  and were older . As men comprised the majority of all patients in severe trauma, at 72.5% of all cases, gender could not be identified as a risk factor , especially in contrast to the non-shoulder group . Focusing on traffic accidents, shoulder injuries were caused significantly more often by motorbike (20.6% vs. 13.4%), bicycle (10.5% vs. 6.8%), or pedestrian accidents (9.6% vs. 8.1%) . Accidents with cars and trucks (26.7% vs. 31.2%), falls < 3 m (9.1% vs. 11.9%) and falls > 3 m (18.8% vs. 20.5%) were less frequently associated with shoulder injuries  . Injuries of the shoulder are less common in patients with abdominal (26.6% vs. 28.4%), pelvic (25.4% vs. 26.6%), or lower limb (35.5% vs. 38.2%) trauma  , head (56.1% vs. 52.2%), and spine (40.5% vs. 36.8%) injuries   occurred significantly more often in combination with a proximal humerus fracture. Whereas lesions of the clavicle (11.0% vs. 12.4%) (p < 0.0001) occurred less often in combination with a proximal humerus fracture , the AC/SC-joint (1.3% vs. 1.2%), nerves (6.6% vs. 0.9%), vessels (1.3% vs. 0.2%), and other (4.2% vs. 2.1%) . In addition, a lower RISC II score was found in patients with shoulder injuries , highlighting the better prognosis of shoulder patients after multiple trauma. Furthermore, shoulder patients showed a more aggravated clinical course than non-shoulder patients, as they had a greater need for red blood cell concentrate (RBC) transfusions  and were more likely to be admitted to the ICU/IMCU . Patients in the shoulder group presented a GCS ≤ 8 less often than non-shoulder patients  , vessel injuries 77.4%), and prox. humeral fractures (74.3%) were most likely to undergo surgical treatment. Clavicle fractures, nerve lesions, scapula fractures, and injuries to the AC/SC-joint were more often treated conservatively Fig. . The rat7.4%, andExtremity injuries have a high prevalence in severe multiple trauma, with 58.6% of multiple trauma patients presenting significant extremity injury. Affection of the clavicle is the third most common fracture in polytraumatized patients (10.4%), only surpassed by femoral (16.5%) and tibial 12.6%) fractures .6% fract.In this context, our findings can be summarized as followed.Show a higher rate of head and thoracic injuries.Suffer significantly more often from pneumothorax and hemothorax.Stay longer on the ICU and in the acute care hospitals.Present a higher ISS than severely injured patients without shoulder injuries.Were likely to have been involved in a traffic accident.Polytraumatized patients (ISS ≥ 16) with shoulder injuries:The high prevalence of shoulder injuries 27.9%) in severely injured patients underlines the importance of understanding the main mechanisms and factors leading to shoulder injuries. Banerjee et al. already showed a 39.6% prevalence of upper extremity injury in polytraumatized patients 7.9% in s and fracAs initially hypothesized, the severity of the injury determines the rate of surgical treatment. Surgical therapy of proximal humerus fractures and injured vessels are very common. In contrast, scapula fractures and clavicle fractures underwent surgical therapy less often. Extraarticular scapula fractures represent approximately two-thirds of all scapula fractures  and are The associated concomitant injuries of shoulder injuries in the shoulder group aggravate the clinical course after severe multiple trauma. They received RBC transfusions, which are known to trigger complications and mortality dose dependence , signifiWe hypothesize that the high correlation of thoracic injuries with shoulder injuries is caused by the anatomical proximity of these structures and, therefore, the thorax is likely to be simultaneously injured in a shoulder injury, and vice versa. Additionally, in the shoulder group we observed numerous motorbike accidents, which are known to increase the risk of thoracic injuries . In seveDue to severe concomitant injuries, our findings underline the importance of an early computed tomography scan (CT-scan) of severely traumatized patients, particularly as suggested by guidelines . AdditioNevertheless, structured and repeated clinical examinations (body check) remain a key aspect in acute trauma care, especially to identify concomitant injuries that may have been missed initially. Further studies showed that brachial plexus injuries may be present in 1% of multiple trauma patients . With 4%Focusing on the ISS and RISC II, shoulder patients presented a significantly increased ISS. Unexpectedly, the RISC II score and the mortality of the study population are lower in the shoulder group, and a contemporaneous increase with the ISS could not be shown. Initially, we assumed a higher RISC II and higher mortality in the shoulder group due to the increased ISS. Previous studies already showed a lower mortality rate in severely injured patients with scapular fractures and aggravated concomitant injuries , 23, butWith regard to the higher ISS in the shoulder group, a previous study showed that a higher ISS could predict lower general health condition with respect to the long-term outcome after trauma . HoweverRegarding the mechanism of injury, traffic injuries have a huge impact on trauma cases worldwide and are the leading cause of death of young adults in Europe , 51. ThiThe present study has limitations, as the analyzed data is retrospective. As the participating hospitals have different levels of patient-centered care, the treatment after trauma could differ. As shoulder injuries could have been initially overlooked, there is a chance that shoulder injuries have been under reported. Furthermore, in polytraumatized patients, minor injuries may be omitted. The choice of diagnostics in the database leading to the diagnosis is not reported, so the accuracy of the data database is dependent on the individual examiner and submission of data in the register. Unfortunately, the exact specification of soft tissue injuries could not be classified.In summary, our findings show that shoulder injuries in severely injured patients are frequently associated with severe concomitant injuries, mainly of the chest, head, and spine. High-speed traffic accidents are the most common injury mechanisms, particularly in motorbike, bicycle, and pedestrian accidents. Previous studies on extremity injuries of the TraumaRegister DGU® confirmed the increase of the ISS, duration of stay, and aggravated clinical course of extremity injuries , 17.Shoulder injuries have an aggravating impact on the clinical course and prolong the clinical stay. As multiple trauma patients with shoulder injury present more diagnoses, the clinical management and treatment of the patient are aggravated. This is demonstrated by a higher initial ISS and longer stay on ICU/IMCU. Nevertheless, an increased rate of mortality in patients with shoulder injuries could not be identified. Our results correspond with empirical data in our daily clinical routine and other national and international findings in the literature. Furthermore, because of the large sample size of the analyzed study population and the high number of included trauma centers providing data, the clinical relevance of our findings seems significant. In the emergency room (ER) and in the tertiary survey, potential shoulder injuries and identified concomitant injuries should be closely considered.


Learning and memory are essential to organism survival and are conserved across various species, especially vertebrates. Cognitive studies involving learning and memory require using appropriate model organisms to translate relevant findings to humans. Zebrafish are becoming increasingly popular as one of the animal models for neurodegenerative diseases due to their low maintenance cost, prolific nature and amenability to genetic manipulation. More importantly, zebrafish exhibit a repertoire of neurobehaviors comparable to humans. In this review, we discuss the forms of learning and memory abilities in zebrafish and the tests used to evaluate the neurobehaviors in this species. In addition, the pharmacological studies that used zebrafish as models to screen for the effects of neuroprotective and neurotoxic compounds on cognitive performance will be summarized here. Lastly, we discuss the challenges and perspectives in establishing zebrafish as a robust model for cognitive research involving learning and memory. Zebrafish are becoming an indispensable model in learning and memory research for screening neuroprotective agents against cognitive impairment. Danio rerio) are tropical fish native to southern Asia and became a vertebrate model organism in developmental biology pioneered by George Streisinger in the 1970s at the University of Oregon. Since then, zebrafish have emerged as one of the animal models in preclinical studies for understanding various physiological processes and diseases. The growing interest is attributed to the favorable features offered by this species. These include ex vivo fertilization, transparent embryos and larvae (facilitate imaging), small size (2–5 cm for adult), easy maintenance, prolific nature (more than 100 eggs produced per fish), rapid development , their accessibility for genetic manipulations such as CRISPR and high homology (70%) to the human genome [Zebrafish  is the most prevalent form of dementia 60–70%) that affects more than 57 million people globally 0% that a,9,10. DeZebrafish show a high degree of conservation in the neuroanatomical organization and neurotransmitter signaling pathways with humans ,11. The Non-human primates and rodents are the classical animal models for behavioral studies involving cognition . Non-humThis review describes the cognitive behaviors related to learning and memory in zebrafish and the assessment tools available for these behaviors. We summarize pharmacological studies that used zebrafish models to screen for the effects of neuroprotective and neurotoxic compounds on cognitive performance. Challenges and perspectives in establishing zebrafish as a robust model to study cognitive impairment are also discussed.Cognition is the mental process of acquiring knowledge and understanding through thinking, learning, memorizing and sensing. The cognitive process includes sensation, perception, motor skills, attention, memory, executive function, language and processing speed . LearninZebrafish display different learning abilities, such as associative, non-associative, social  and motor learning. In social learning, a group of zebrafish learned faster than a single individual . In motoNon-associative learning is a general lasting change in response strength toward a stimulus due to repeated exposure. Non-associative learning is further categorized into habituation and sensitization. Habituation is the decrease while sensitization is the increase in the animal’s response to a sensory stimulus upon continuous exposure. The changes in responses could be in the short- and long-term. Habituation has been regarded as an evolutionarily conserved behavior for optimal survival. Habituation allows the animal to disregard repeated stimuli while focusing on important stimuli such as potential predators and danger in the environment ,19. In tAssociative learning is the process of acquiring new information by linking two elements. This type of learning is more commonly assessed than non-associative learning in zebrafish studies. The two forms of associative learning are classical (Pavlovian) and operant  conditioning ,16,27. IIn operant conditioning, an animal learns to correlate its voluntary behavioral responses with their consequences . The terClassical conditioning can be further divided into appetitive and aversive conditioning based on the nature of the US. Appetitive conditioning utilizes favorable stimuli as the US. Sison and Gerlai demonstrated that zebrafish are capable of associative learning by using food as the reward and a red card as the visual cue . After tIn aversive conditioning, the CS is paired with a fear-inducing US that leads to a fear-related conditioned response when the CS is administered alone. The nature of the CS can be contextual (location or environment) or cued . The most commonly used aversive US in zebrafish studies is the application of electric shock (ES). Fear conditioning is the most common form of classical learning assessed in zebrafish. Valente and colleagues demonstrated that both larval and adult zebrafish could perform cued fear conditioning in a paradigm that associated a checkerboard pattern (CS) with the ES (US) . After tIn contextual fear conditioning, a novel environment is associated with the US. Kenney and colleagues showed that contextual fear conditioning in zebrafish lasted for at least 14 days and varied in the fear extinction rates among different strains . ExtinctFear-related responses include freezing (immobility), erratic movements (zigzagging), bottom-dwelling, a tighter shoal, leaping (jumping) and thigmotaxis . In the Operant conditioning can be classified as reinforcement or punishment, such that the paradigm increases or decreases the response to modify the strength of the response over the course of training. The reinforcement in operant conditioning can be positive (addition of reward as a reinforcer) or negative . Both positive and negative reinforcement paradigms are aimed at strengthening the behavioral response.Positive reinforcement administers the appetitive US after the animal performs a behavioral response to strengthen the response. Manabe and co-workers developed an automated operant device for positive reinforcement conditioning in zebrafish. The response key was equipped with a sensor that dispenses brine shrimp eggs when it was approached by zebrafish . In the Negative reinforcement removes the aversive US when the animal engages in a behavioral response. This type of conditioning can be further divided into escape learning and avoidance (active and passive). Escape learning refers to the animal engages a behavior to terminate the ongoing aversive stimulus. For example, the crossing response of rodents will be increased from a compartment with existing foot shock to the opposite compartment in a shuttle box. The active avoidance paradigm requires the presentation of a cue  prior to the administration of an aversive stimulus. Active avoidance was performed to prevent the happening of the aversive stimulus. Xu and colleagues demonstrated that zebrafish increased crossing response to a compartment without ES upon presenting a light signal previously paired with the administration of ES . In passSimilarly, punishment-based operant conditioning is divided into positive (impose punishment) and negative (remove appetitive stimulus as punishment). Both positive and negative punishment paradigms are aimed at weakening the behavioral response. For positive punishment, an aversive US is administered when a behavioral response is engaged, eventually leading to a decreased behavioral response. The difference between positive punishment, escape learning and avoidance is that the behavior response in positive punishment leads to the administration of an aversive stimulus, while the behavioral response in the latter two negative reinforcement results in the removal/prevention of an aversive stimulus. For example, in positive punishment conditioning, a rodent learns to refrain from pressing a lever that will lead to foot shock.For negative punishment, an appetitive US is removed when the animal performs a behavioral response. For instance, this conditioning is used in dog training to correct undesired behavior by taking away the reward (food or toy). To our knowledge, both positive and negative punishments have not been engaged in pharmacological studies using zebrafish as models to assess cognitive performance.In summary, zebrafish are capable of performing both non-associative and associative learning tasks. This allows the researcher to assess the zebrafish’s cognitive performance using various tests. From a survival perspective, operant learning allows the animals to find a safe and rewarding outcome while avoiding danger in a complex environment. Unsurprisingly, operant conditioning is pervasive in zebrafish studies because task performance could be enhanced by adding an operant component . NeverthMemory can be classified mainly into sensory, short-term and long-term ,37. SensNot all forms of memory can be evaluated in zebrafish as model organisms. Similar to rodent models, the most common types of memory assessed in zebrafish for cognitive studies are spatial, recognition and associative memory. To score the memory retention index, a time delay between the training (familiarization) and test (probe) phases is required. Spatial memory in zebrafish is assessed based on the preference of zebrafish for new space/area. The cue to a novel place could be contextual or visual . Cognato et al. showed that spatial memory in zebrafish lasted up to 3 h after the familiarization session and diminished after 6 h of exposure . ObjectiThe main types of behaviors assessed in zebrafish as pharmacological models for learning and memory performance are locomotion, emotion (anxiety-like), cognition  and social interaction  . Table  There coThe locomotor activity test (LAT) is used to evaluate the spontaneous swimming behaviors of zebrafish . It is nLAT is generally performed first in a battery of behavioral tests before other more complicated paradigms. This can prevent the animals from becoming exhausted and less motivated to perform the subsequent paradigms. To our knowledge, there is no consensus on a standardized environment or environmental conditions for LAT. Most behavioral tests developed for zebrafish serve as a general guide rather than a standardized protocol. Considering that zebrafish are increasingly used as study models across different laboratories worldwide, standardization may be necessary to make the findings from different studies comparable. Recently, Maeda et al. developed a standardized method for assessing the behavioral response (including locomotor activity/response) in zebrafish larvae (6 to 7 dpf) using a light–dark locomotion test . The chaA novel tank test is, similar to the LAT setup, used to assess the anxiety-like behavior of zebrafish . The tanThe inhibitory avoidance test (IAT) is one of the most commonly used cognitive tests to evaluate the learning and memory functions of zebrafish. The test can be established using a two-chamber tank A and a mSimilar to IAT, a rectangle tank or maze is used to establish the CS–US pairing over repeated exposure. Chen et al. covered the left and right arms of a T-maze with green and red colored sleeves as visual cues . One of The Y-maze test is used to inspect the spatial memory of zebrafish based on their innate behavior that tends to explore the novel place . ZebrafiSimilar to the Y-maze that is based on the fish response to novelty, a novel object recognition test is used to examine the recognition memory. In the test conducted by Capatina et al., a zebrafish was placed in a tank with two familiar objects (red cubes) and allowed to explore the tank for 10 min . After aThe neurobehavioral tests for zebrafish have been reported for over two decades . The utiOriganum vulgare spp. hirtum (Lamiaceae)) essential oil on the scopolamine-induced amnesia zebrafish model [Juglans regia L.), on the scopolamine-induced cognitive deficit zebrafish model [Cholinergic signaling is involved in memory acquisition and consolidation. Reduction of acetylcholine (ACh) due to hydrolysis by acetylcholinesterase (AChE) could implicate the development of dementia and AD. Scopolamine is an alkaloid that acts as a nonselective muscarinic receptor antagonist. It blocks the receptors to induce cognitive impairment. Scopolamine-induced amnesia in zebrafish is a widely used model for screening drugs with neuroprotective effects . Capatinsh model . Scopolash model . The autsh model , lithiumsh model , sulforash model , adenosish model , flavonosh model , and physh model .Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 [Lactobacillus plantarum ST-III using the triclosan-induced neurodegeneration zebrafish model [Lim et al. used a high-cholesterol diet-induced cognitive impairment model of zebrafish to explore the effect of two lactic acid bacteria (LAB) strains: um LAB12 . The fissh model . They prEmbelia ribes), on acute and chronic PTZ-induced seizures and cognitive impairment. In the acute seizure model, pre-treatment of embelin (0.078–0.625 mg/kg) reduced seizures and ameliorated epilepsy-associated cognitive dysfunction induced by a single dose (170 mg/kg) of PTZ [ccl2, tlr-4, tnf-α, il-1, ifn-γ).Kundap et al. developed a zebrafish model of epilepsy that induced seizure-like behaviors and cognitive dysfunction using pentylenetetrazole (PTZ) . The mod) of PTZ . Time sp) of PTZ . Daily pQuercetin has neuroprotective properties but is limited with lower bioavailability and permeability across the blood–brain barrier. Rishitha and Muthiraman studied the efficacy of nanoparticle-formulated quercetin on PTZ-induced cognitive impairment in zebrafish . IntrapeN-Methyl-D-aspartate receptors are ionotropic glutamate receptors related to cognitive functions. MK-801, an N-Methyl-D-aspartate receptor antagonist, has been shown to induce amnesia in zebrafish ,82,83. OAlcohol consumption in higher doses can impair cognitive functions leading to amnesia or blackout. Rapid elevated ethanol levels impair memory consolidation, resulting in memory gaps for individuals during intoxication. Bertoncello et al. developed a novel ethanol-induced amnesia model in zebrafish to evaluate the effect of taurine on memory consolidation . The aniA receptors. Using the hypoxic zebrafish model, the same research group showed that magnesium sulfate prevented cognitive deficits and brain infarction via the up-regulation of EET4A glutamate receptor [Hypoxia results in ischemic injuries to neurons and cognitive deficits. It inhibits glutamate uptake into the neurons. Urinary trypsin inhibitor (UTI) is a urinary glycoprotein that inhibits proteolytic enzymes such as hyaluronidase plasmin, α-chymotrypsin and trypsin. Kim et al. demonstrated the neuroprotective effect of UTI on a cognitively impaired zebrafish model induced by hypoxia . UTI-trereceptor .c-fos and bdnf in the zebrafish brain. Pusceddu et al. utilized zebrafish to evaluate the chronic effects of Mediterranean natural extracts, namely licorice (Glycyrrhiza glabra) root extract and rosemary extract, on cognitive functions [Healthy and cognitively unimpaired zebrafish are models for screening compounds or conditions with memory enhancement (nootropic) properties ,88,89,90unctions . Both exunctions . Methyleunctions . Braida unctions . They foZebrafish are a popular model used to screen for compounds with neurotoxicity effects on learning and memory. These include metals/elements ,100,101,Shang et al. used zebrafish to validate the neurotoxic mechanism of occupational aluminum exposure in humans . Serum a2) reduced the fish locomotor activity and aggressive behaviors in a 3D LAT and mirror biting test, respectively. In the mirror biting test, a mirror was located next to the tank. The aggressive behavior was scored based on biting and fast swimming, as zebrafish tend to display boldness by biting the mirror and tracing their reflection by quick movement. The PbCl2 induced anxiety-like behaviors in a novel tank test and altered circadian rhythm locomotor activity. Fish exposed to PbCl2 had reduced latency to enter the dark compartment with ES in the IAT, indicating memory loss induced by PbCl2 poisoning. Elevation of cortisol and reduction of serotonin and melatonin in the brain could be related to the altered neurobehaviors observed in PbCl2-exposed zebrafish. Sarasamma et al. developed an AD model based on zinc chloride (ZnCl2)-induced cognitive impairment in zebrafish [2 decreased the latency to enter the dark chamber with ES in an IAT for trained fish, signifying lower memory retention. Elevation of reactive oxygen species level, lipid peroxidation markers  and stress hormones (catecholamine and cortisol) and reduction of melatonin and antioxidant enzyme activities  were observed in the brain following ZnCl2 exposure. The compound also affected neurotransmitters by increasing AChE activity, dopamine, glutamate and GABA levels, while decreasing glycine and histidine levels. More importantly, ZnCl2-exposed zebrafish displayed AD-like symptoms by elevating amyloid beta 42 and phosphorylated tau proteins in the brain.Bui Thi et al. utilized zebrafish to evaluate the effect of chronic lead exposure on neurobehaviors . Low levebrafish . ZnCl2 dAudira et al. investigated the adverse effect of chronic exposure to donepezil on cognitive functions and behaviors, using normal zebrafish as a model to represent healthy individuals without AD . Long-teZebrafish larvae can be used to perform toxicity screening on learning and memory. He et al. used zebrafish larvae to investigate the effect of prenatal exposure to propofol, a widely used general anesthetic drug, on learning and memory function . PropofoThere are several concerns when using zebrafish as models for drug discovery in cognitive research. First, aqueous immersion was the major route of drug administration used in many studies, which might be least applicable to humans. Aqueous immersion was chosen probably due to the convenient setup. However, the bioavailability of the drugs is difficult to assess in zebrafish via this method. Over the years, other routes of administration, such as intraperitoneal ,67,122, In this review, we have seen that zebrafish have emerged as one of the vertebrate models for cognitive research related to learning and memory functions over the past decade. The gaining popularity is supported by a repertoire of neurobehavioral displayed by the zebrafish and assessment tools available for these behaviors. Zebrafish have been proven useful as pharmacological models in drug screening for neuroprotective or neurotoxicity agents that affect learning and memory performance. Zebrafish emerged as a complementary model to rodents in preclinical studies before further testing on humans. One of the main advantages of the zebrafish model is lower maintenance costs for pharmacological screening. However, the clinically relevant routes of drug administration still require further exploration. In addition, innovative behavioral assessment apparatuses and software are needed to increase the assay throughput. By overcoming these limitations and combining the favorable features of zebrafish, this animal model will be indispensable in drug discovery research involving learning and memory.


Serological testing for SARS-CoV-2 IgG antibodies is used to assess their presence in blood samples from exposed individuals and provides a measure of the magnitude of immune response to infection. The measurement of neutralizing antibodies (NAbs) in particular provides information about the severity of prior infection and level of protective immunity against re-infection. Much of the work investigating the association between prior infection severity and NAb levels has been conducted among clinical populations, and less is known about this relationship in the general population. Accordingly, we utilize data from a large (n = 790) community-based cohort of unvaccinated, seropositive participants. We analyzed the association between NAb response, measured via surrogate virus neutralization assay, with patterns of symptoms and household exposure. Our results indicate no detectable NAb activity in 63.8% of the seropositive participants (n = 504). Those with detectable NAb levels demonstrated a positive relationship between NAb activity and both self-reported previous symptom severity and household exposure. These findings are significant in light of recent concerns about degree of protective immunity conferred by prior infection or vaccination, and we highlight the value of community-based research for investigating variation in immune response. Response to infection with SARS-CoV-2 can be highly variable, including differential degrees of symptom severity, hospitalization, and immune response. Serological testing has been a vital tool in tracking SARS-CoV-2 infection across communities, as it is used to detect the presence of antibodies to SARS-CoV-2 in blood samples from exposed individuals, while also providing a measure of the magnitude of immune response to infection4.As of July 22, 2021, more than 190 million people worldwide have been infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19)6. NAbs are effective at inhibiting this initial interaction, as well as downstream events, and thereby preventing viral entry into cells5. Following infection, NAbs have been shown to persist for many months, albeit with decreasing levels detectable in the blood8.Serological testing facilitates assessment of exposure and immune response via laboratory measurement of antibodies that impede infection by SARS-CoV-2. Some, but not all, antibodies directed against the spike protein of SARS-CoV-2 can neutralize virus infectivity in laboratory assays. These neutralizing antibodies (NAbs) bind to the SARS-CoV-2 surface spike protein on the virion surface, the part of the virus that engages the human angiotensin-converting enzyme 2 (ACE2) receptor in order to gain entry into host cells9. Similarly, COVID-19 patients who were treated in an intensive care unit demonstrated significantly higher peak neutralizing antibody titers compared with those who were not11. Further, higher neutralizing antibody levels among vaccinated individuals have been shown to predict a lower likelihood of SARS-CoV-2 breakthrough infections11. These results point to a consistent relationship between disease severity and level of NAb activity in clinical populations.Because of their key role in hindering viral entry, it is expected that neutralizing antibody activity can provide information about severity of prior infection and the level of protective immunity against re-infection. This expectation is supported by results indicating that NAbs appeared earlier and at higher levels in patients who experienced severe or moderate infections, when compared with patients who experienced mild or asymptomatic illness12. We utilized data from a large community-based cohort in Chicago in order to investigate these dynamics in a non-clinical population that includes people who did require treatment or even know they had been infected with SARS-CoV-2. Because the majority of COVID-19 cases are asymptomatic or mild13, our community-based sample adds a clearer picture of the nature of the association between disease severity and the production of NAbs, which likely provides at least partial protection against re-infection in the broader population of all previously infected and unvaccinated persons.Less is known about this relationship in the general population. Increasing our understanding of variation in NAb levels across severity of infection in the community is particularly critical as many people remain hesitant to receive the SARS-CoV-2 vaccine, based on beliefs that any prior exposure will provide adequate protection against re-infection by the virus15. Previous work also indicated that household exposure to the virus, which is likely greater or more prolonged than exposure outside the home, is associated with both greater disease severity and higher antibody concentrations15. However, it is not yet known whether there is a similar association between the level of NAb activity and either COVID-19 symptom severity or household exposure history in a non-clinical population.Previous work by our team using data from a community-based observational study in Chicago has shown that those who reported more symptoms of infection in months prior to antibody quantitation had higher concentrations of immunoglobulin G (IgG) antibodies directed against SARS-CoV-2 spike receptor binding domain (RBD)Here, we analyze the association of magnitude of NAb response, using a surrogate virus neutralization assay, with patterns of symptoms and household exposure history among unvaccinated, seropositive participants. Our results indicate that the majority of individuals who tested seropositive for prior COVID-19 infection had no detectable neutralization activity. In individuals who had detectable levels of neutralizing antibodies, we report a positive association between COVID-19 symptoms and neutralization activity. We also report higher neutralization activity in individuals living with household members who reported symptoms or diagnosis of COVID-19.4. The study included a total of 4463 adults at this time point.A large community-based sample of adults living within the Chicago, IL metropolitan area was recruited to participate in a study called Screening for Coronavirus Antibodies in Neighborhoods (SCAN). Samples for this analysis were collected between June 24 and November 11, 2020, prior to availability of SARS-CoV-2 vaccination in the areaParticipation was facilitated by a web-based, “no contact” protocol, which allowed individuals to provide information and dried blood spot (DBS) samples outside of a clinical setting. Participants were recruited via advertisements in social media, emails, print flyers, newspapers, participant registries, participant referrals, community outreach, and local press. Recruitment was conducted in neighborhoods throughout the Chicago metropolitan area and at the Northwestern University Feinberg School of Medicine (FSM) in Chicago. To ensure racial and gender diversity and representation within the sample, we carried out stratified random sampling to adaptively match enrollment of white participants and women (groups that were more likely to complete the screener) to enrollment of non-white participants and men.17. All research activities were implemented under protocols approved by the institutional review board at Northwestern University (#STU00212457 and #STU00212472). Written informed consent was received from all individuals prior to participation in this study, and all methods were performed in accordance with relevant guidelines and regulations.Study data were collected and managed using REDCap electronic data capture tools hosted at Northwestern University2), smoking, number of individuals in their households, and whether any cohabitants had been diagnosed with COVID-19 or had symptoms of COVID-19.Participants provided information about COVID-19 testing, diagnoses, and symptoms experienced after March 1, 2020. Other variables included sex (based on assignment at birth), self-identified racial/ethnic identity, pre-existing chronic medical conditions  and the timing of blood collection (Supplementary Table 18. IgG antibodies were quantified using an enzyme-linked immunosorbent assay (ELISA) that has received emergency use authorization from the United States Food and Drug Administration . This assay was adapted and validated for use with DBS samples18.Analyses presented here focus on the 17.7% (n = 790) of the 4463 total participants that tested seropositive for prior infection based on the presence of IgG antibodies against the RBD of SARS-CoV-215. The symptoms not included in the cluster of eight are itchy eyes; runny nose; and sore throat.Prior to DBS collection, participants indicated whether they had experienced any of the following eleven symptoms potentially associated with COVID-19: headache; fatigue or excessive sleepiness; sore throat; cough; muscle or body aches; runny nose; fever or chills; diarrhea, nausea, or vomiting; shortness of breath; loss of sense of smell or taste; and itchy eyes. In a previous study, we identified a cluster of eight of these symptoms that were associated with higher SARS-CoV-2 IgG concentrations: headache; fatigue or excessive sleepiness; cough; muscle or body aches; fever or chills; diarrhea, nausea, or vomiting; shortness of breath; loss of sense of smell or taste15). Briefly, each of the eight symptoms associated with higher SARS-CoV-2 IgG antibody levels was weighted by the regression coefficient that resulted from a bivariate model that included the symptom as the independent variable and log10-transformed SARS-CoV-2 IgG concentration as the dependent variable. For the symptom score, our goal was to establish the independent association between each individual symptom and SARS-CoV-2 IgG antibody levels—which was facilitated by using bivariate models (that did not include other symptoms). Because participants reported distinct combinations of symptoms , we did not seek to create scores that included the correlation between symptoms or their mutual influence, as doing so might under-or over-estimate the significance of the individual symptom. The resulting symptom weights are: fever = 0.22, cough = 0.13, shortness of breath = 0.20, headache = 0.09, muscle or body aches = 0.19, fatigue or excessive sleepiness = 0.13, diarrhea/nausea/vomiting = 0.17, loss of taste/smell = 0.3215. This composite variable indicated how severely each participant experienced symptoms associated with COVID-19 infection.A “COVID-19 symptom severity score” for each participant was created following methods described previously  protocol . This method, which can be implemented with immunoassay techniques, contrasts with other conventional methods for measuring neutralizing activity that require the presence of live virus and specialized laboratory containment facilities. Excellent concordance between results of these types of assays of NAb activity has been reported20. Based on these validation results, the threshold for determining the presence of surrogate neutralization activity was set at 13.2% or higher20.The sVNT method was adapted and validated to measure neutralizing antibodies in DBS samples described here21.Patterns of association between symptoms and the presence of NAb were established using summary statistics and multiple linear regression analyses. Due to the skewed distribution of NAb values, median values are presented as descriptive statistics and log10-transformed values were used for regression analyses. Covariates in the multiple linear regression analyses included age, race/ethnicity, sex assigned at birth, and chronic pre-existing conditions. All data analyses were conducted using R (version 4.0.4) in RStudio Version 1.4.1106Table Of the seropositive participants included in these analyses, 67.7% of participants reported experiencing one or more of eleven COVID-19 symptoms listed above since March 1, 2020. Notably 32.3% reported no prior symptoms.2 = 0.12; p < 0.001)  (Table Seropositive participants (n = 790) had a mean symptom severity score of 0.32 . COVID-19 symptom severity scores were higher among participants with detectable NAb activity than in participants with no neutralization activity . The median symptom severity score for participants with neutralization activity was 1.16, and median score for participants without detectable neutralization activity was 0.30. A linear regression model adjusted for the same covariates demonstrated a positive linear association between surrogate neutralization levels and COVID-19 symptom severity score . Similarly, participants with neutralization activity were also more likely to have a household member who reported one of the 11 symptoms potentially indicative of COVID-19 . A regression model adjusted for age, birth sex, race, and preexisting chronic medical conditions demonstrated that living with someone previously diagnosed with COVID-19 or having a household member with symptoms potentially indicative of COVID-19 was positively associated with levels of surrogate neutralization activity  . Symptom severity score of the study participant remained positively associated with surrogate neutralization levels in this model .Individuals with neutralization activity were more likely to report a household member who had been diagnosed with COVID-19 than participants without detectable levels of NAb activity (23.4% vs. 4.8%; χOur results indicate a positive relationship between neutralizing antibody activity and both self-reported previous symptom severity and household exposure to the virus, in a community-based sample of unvaccinated, seropositive persons. We also found no detectable surrogate neutralization activity in the majority of seropositive individuals, indicating that lightly symptomatic or asymptomatic SARS-CoV-2 infection does not elicit a strong NAb response.24. Our results are consistent with findings from clinical populations that indicate that more severe symptomatic cases of COVID-19 are associated with higher NAb levels, suggesting that these cases are more likely to provide increased and longer-lasting protective immunity following infection25.These results enhance our understanding of the variation in NAb levels across individuals who have different responses to exposure to SARS-CoV-2. These results are particularly significant in light of recent concerns about the persistence of both total antibodies and NAbs to SARS-CoV-2 following infection, particularly in those with mild or asymptomatic cases, and the degree of protective immunity conferred by prior natural infection26. Results such as these support previous findings indicating that prior SARS-CoV-2 infection, especially when mild or asymptomatic, may not be a reliable indicator of ‘natural immunity’ from reinfection, and consistent with the expectation that many previously infected persons are likely to have a relatively low level of protection from reinfection25. These results are consistent with our previous findings that two doses of the mRNA vaccine were required for individuals who had mild/asymptomatic seropositive cases to attain a level of surrogate neutralizing antibody response comparable to individuals who had previously been diagnosed with COVID-1927. Taken together, these findings suggest that natural infection—particularly mild/asymptomatic seropositive cases—are most likely to provide a level of immune protection comparable to one dose of mRNA vaccine29. Further, our results indicate that, to clinically assess the potential protection conferred from previous infection, it will be helpful to assess symptom severity and method of exposure to the virus, as a surrogate for NAb activity.However, our results also highlight that the majority of seropositive individuals in this community-based study did not exhibit neutralization activity. These findings are consistent with other studies conducted with non-clinical populations that have reported a low proportion of seropositive individuals with detectable NAbs30. In addition, completion of two mRNA vaccine doses was reported to elicit a broader NAb response better covering all variants of concern than did full vaccination of previously uninfected persons31. However, it is not yet known whether previously infected individuals with varying levels of disease severity differ in their antibody responses that may impact vaccine effectiveness, potential for onward transmission, including for more transmissible Delta variants and other variants of concern that may emerge in the future, after the full course of mRNA vaccinations. Because samples collection for this study predated wide-scale vaccination efforts and the emergence of the Delta variant, we are not able to test these questions directly. Nevertheless, the results of our study highlight the value of community-based research including the full spectrum of SARS-CoV-2 infection for investigating variation in immune response across infected individuals with differential symptomatology. Continuing to examine these dynamics with attention to symptom severity, as well as vaccination history and vaccination responses, can further inform public health strategies to control impacts of SARS-CoV-2 infection.Our findings are also relevant to questions concerning the degree of immune protection experienced by individuals who have received a COVID-19 vaccination following SARS-CoV-2 infection. Individuals with previous symptomatic or asymptomatic COVID-19 infection have been shown to have higher neutralizing antibody titer responses to a single dose of mRNA vaccine than those who were not previously infectedSupplementary Information.