Long non-coding RNAs have been shown to be associated with cancer development and progression, demonstrating potential applications as novel diagnostic or prognostic molecular markers in clinical management and treatment. However, the functional significance of lncRNAs in the development and malignant progression of gliomas is still unclear and needed to be further explored. we first obtained genome-wide lncRNA expression profiles in a large cohort of patients with gliomas from the Gene Expression Omnibus database using microarray probes repurposing method and investigated the lncRNA expression patterns during the tumorigenesis and malignant progression of gliomas. By using differential expression analysis, we identified a large number of lncRNAs that were associated with the tumorigenesis and malignant progression of gliomas in the training dataset and showed their robustness in the testing dataset. Subsequently, we identified a novel four-lncRNA signature which was closely related to the prognosis of patients with GBM. The prognostic value of this signature was verified in the test set of 80 patients. Functional analysis suggested that the four lncRNAs associated with survival of patients with GBM may be involved in cancer-related biological processes and pathways and their deregulation may lead to GBM tumorigenesis and progression. These novel lncRNA biomarkers will improve our understanding of the molecular mechanisms during the development and progression of glioma and provide novel diagnostic or prognostic markers and therapeutic targets for gliomas. Gliomas are the most common type of primary brain tumor, accounting for 80% of all malignant brain tumors and 30% of all brain and central nervous system tumors. Gliomas are further categorized into low-grade gliomas (WHO grade I and II) and high-grade gliomas (WHO grade III and IV) according to their histopathologic characteristics such as cytological atypia, anaplasia, mitotic activity, microvascular proliferation, and necrosis . The medDuring the past decades of RNA biology, a novel class of RNAs, termed long noncoding RNAs (lncRNAs), has been identified as an important layer of genome complexity . lncRNAsThe purpose of this study is to identify specific lncRNA markers significantly associated with the tumorigenesis and malignant progression of gliomas by studying differential lncRNA expression pattern in glioma and normal brain tissue or in different WHO grades in a large cohort of patients with gliomas from the Gene Expression Omnibus (GEO), and identify a lncRNA signature which could act as a prognostic predictor for patients with glioblastoma multiforme (GBM).To identify potential lncRNAs associated with glioma development, we performed differential expression analysis for lncRNAs between normal brain tissues (n=8) and glioma samples (n=276) using significant analysis of microarray (SAM) method in the training dataset. In total, we identified 299 lncRNAs that are differentially expressed in normal brain tissues compared to glioma samples using SAM with an adjusted P-value <0.01 after Benjamini & Hochberg correction and fold change > 2.0 (or <0.5). Of these, 133 lncRNAs were over-expressed and 166 lncRNAs were down-regulated in glioma samples compared to normal brain tissues. Hierarchical clustering of all samples in the training dataset according to the expression value of these 299 differentially expressed lncRNAs based on centred Pearson correlation clearly separated glioma samples from normal brain tissues  and high-grade gliomas  using SAM method in the training dataset. In total, we identified 47 lncRNAs that are differentially expressed between low-grade gliomas and high-grade gliomas using SAM with an adjusted P-value <0.01 after Benjamini & Hochberg correction and fold change > 2.0 (or <0.5). Of these, 18 lncRNAs were over-expressed and 29 lncRNAs were down-regulated in high-grade gliomas compared to low-grade gliomas. We clustered the glioma samples in the training dataset according to the expression values of 47 differentially expressed lncRNAs between low-grade gliomas and high-grade gliomas and obtained two distinctive sample clusters: cluster 1 containing 28 out of 32 low-grade gliomas and 198 of 244 high-grade gliomas with an 88.41% accuracy Figure . StatistFor the predicted progression-related lncRNAs markers, we have examined their expression patterns with respect to grades in the independent GSE4290 dataset by using hierarchical clustering and Chi-square test. As shown in Figure AK098425, AL833059, AK056155 and CR613436) significantly associated with the outcome of patients with GBM. We then used these four lncRNA biomarkers to construct an expression signature by the risk score method as the classifier for prognosis prediction. This four-lncRNA expression signature was defined as a linear combination of the expression levels of the four lncRNA biomarkers and the multivariate Cox regression coefficient as the weight as follows: Risk score=++ +. The risk score was calculated for each patient in the training dataset. Then patients of training dataset were assigned into the high-risk group (n=76) and low-risk group (n=79) using the median risk score as risk cutoff value (0.27). Survival analysis suggested that there is a significant difference in overall survival between high-risk group and low-risk group   Figure .To evaluate the robustness of four lncRNA biomarkers, the four-lncRNA signature was further validated in another independent testing dataset (GSE7696 dataset). By using the same risk score model and cutoff value from the training dataset, 80 patients in the GSE7696 dataset were divided into the high-risk group (n=25) and low-risk group (n=55). Inconsistent with the findings described above, patients in high-risk group and low-risk group showed marginally significantly different overall survival  Figure . PatientIn order to gain an initial understanding of functional roles of this four-lncRNA signature, we first calculated the expression correlation between one of four lncRNAs and protein-coding genes in the training dataset and identified 664 protein-coding genes that are positively correlated with at least one of the four prognostic lncRNAs (top 1%). Then we performed functional enrichment analysis for protein-coding genes at GO and KEGG levels to infer lncRNA function. Results of GO analysis suggested that these 664 protein-coding genes enriched significantly in immune-related biological processes, including inflammatory response, response to wounding, inflammatory response, response to wounding, defense response, immune response, innate immune response, activation of plasma proteins involved in acute inflammatory response, B cell mediated immunity, acute inflammatory response and positive regulation of immune response Figure . ResultsAlthough traditional histopathologic classification is well established based on histological features, histological classifications did not well reflect clinical characteristics and prognosis of patients with grade II-III glioma and glioblastoma (grade IV glioma) , 15. WitMore recently, an extensive amount of functional non-coding RNAs has been discovered by experimental or computational approaches. Many of them have been proved to play important roles in both oncogenic and tumor suppressive pathways . AberranIn our study, we first obtained genome-wide lncRNA expression profiles in a large cohort of patients with gliomas from the Gene Expression Omnibus database using microarray probes repurposing method, and investigated the functional roles of lncRNAs in the tumorigenesis and malignant progression of gliomas by examining expression pattern of lncRNAs in glioma and normal brain tissue or different WHO grades. By using differential expression analysis, we identified a large number of lncRNAs that were associated with the tumorigenesis and malignant progression of gliomas in the training dataset and showed their robustness in the testing dataset. Our results demonstrated that dysregulated lncRNA expression played important roles in the tumorigenesis and malignant progression of gliomas. Hierarchical clustering analysis revealed that lncRNA expression pattern was significantly correlated with disease status. Finally, we identified a novel four-lncRNA signature which was closely related to the prognosis of patients with GBM. The prognostic value of this signature was verified in the test set of 80 patients.In recent years, an increasing number of lncRNAs have been identified. However, the functional study of lncRNAs is relatively slow and only a fraction of lncRNAs was well functionally characterized. It has been shown that lncRNA function could be inferred by studying the functional roles of protein-coding genes that are coexpressed with lncRNAs. Therefore, we performed a preliminary functional study for these four prognostic lncRNAs by performing functional enrichment analysis for co-expressed protein-coding genes. Results of functional enrichment analysis suggested that genes co-expressed with four prognostic lncRNAs tended to be clustered most significantly in nine immune-related GO biological processes and four KEGG biological pathways. Previous studies have demonstrated the close association between immune response and outcome in GBM , 35. LysIn conclusion, we investigated the lncRNA expression patterns during the tumorigenesis and malignant progression of gliomas and their effects on patients’ disease status. Our study has shown that the lncRNA expression pattern is altered during the tumorigenesis and malignant progression of gliomas which can serve as novel molecular markers for diagnosis and prognosis prediction. Using lncRNA expression profiling in Cox proportional hazard regression analysis, we developed a novel four-lncRNA signature that accurately predicts survival in patients with GBM. These novel lncRNA biomarkers will improve our understanding of the molecular mechanisms during the development and progression of glioma and provide novel diagnosis or prognosis markers and therapeutic targets for gliomas.http://www.ncbi.nlm.nih.gov/geo/). The dataset GSE16011 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16011) was selected as our training dataset because it is the largest dataset. Another two datasets GSE4290 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE4290) and GSE7696 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7696) were used as testing datasets. The training dataset contained 276 glioma samples with overall survival information, including 8 grade I sample, 24 grade II samples, 85 grade III samples and 159 grade IV samples, as well as 8 non-tumoral brain tissue controls. The testing GSE4290 dataset contained 157 glioma samples without overall survival information, including 45 grade II samples, 31 grade III samples and 77 grade IV samples, as well as 23 non-tumoral brain tissue controls. Another testing GSE7696 dataset contained 80 glioblastoma multiforme (GBM) samples with overall survival information and 4 non-tumoral brain tissue controls.Patients and normal samples used in this study were from three independent datasets in the Gene Expression Omnibus database  algorithm for background adjustment [64] and log-transformed (base 2). LncRNA expression profiles of CC patients in this study were obtained by repurposing the probes in the HG-U133_Plus_2.0 array according to Zhang's study . BrieflySignificance analysis of microarrays (SAM) was applied to identify differentially expressed lncRNAs between normal brain tissues and glioma samples, and between low-grade gliomas (grade I and II) and high-grade gliomas (grade III and IV). Those lncRNAs with FDR ≤ 0.01(Benjamini and Hochberg's multiple test adjustment) and fold change > 2.0 (or <0.5) from SAM analysis were identified as differentially expressed lncRNAs. Unsupervised hierarchical clustering was used to investigate the effectiveness of lncRNA biomarkers in distinguishing normal brain tissues and glioma samples or low-grade gliomas (grade I and II) and high-grade gliomas (grade III and IV), and the Chi-square test was used to test the significance of the association between sample status and lncRNA biomarkers.To identify novel lncRNAs significantly associated with overall survival of patients with GBM, the univariate and multivariate Cox regression analysis were performed to evaluate the association between each of dysregulated lncRNAs and patient's overall survival. Then a lncRNA signature was developed as a linear combination of the expression value of lncRNAs weighted by their respective multivariate Cox regression coefficients. According to the lncRNA signature, patients were classified into high-risk group and low-risk group using the median risk score from the training dataset as the cutoff. Kaplan-Meier survival curves and log-rank tests were used to assess the differences in survival time between the predicted early-stage-like group and advanced-stage-like patients. Time-dependent ROC analysis was performed to compare the sensitivity and specificity of the survival prediction based on the lncRNA risk score. All analyses were performed using R/Bioconductor.


Different strategies have been developed using Independent Component Analysis (ICA) to automatically de-noise fMRI data, either focusing on removing only certain components  or using more complex classifiers to remove multiple types of noise components . However, denoising data obtained in an acute setting might prove challenging: the presence of multiple noise sources may not allow focused strategies to clean the data enough and the heterogeneity in the data may be so great to critically undermine complex approaches. The purpose of this study was to explore what automated ICA based approach would better cope with these limitations when cleaning fMRI data obtained from acute stroke patients.The performance of a focused classifier (ICA-AROMA) and a complex classifier (FIX) approaches were compared using data obtained from twenty consecutive acute lacunar stroke patients using metrics determining RSN identification, RSN reproducibility, changes in the BOLD variance, differences in the estimation of functional connectivity and loss of temporal degrees of freedom.p < 0.001), localized reduction in BOLD variance consistent with noise removal, and significant changes in functional connectivity (p < 0.001). Patient-trained FIX resulted in higher RSN identifiability (p < 0.001) and wider changes both in the BOLD variance and in functional connectivity compared to ICA-AROMA.The use of generic-trained FIX resulted in misclassification of components and significant loss of signal (< 80%), and was not explored further. Both ICA-AROMA and patient-trained FIX based denoising approaches resulted in significantly improved RSN reproducibility  are tested on acute stroke fMRI data.•Both FIX and AROMA significantly increased RSNs reproducibility.•Their use led to changes in functional connectivity estimates and BOLD signal variance.•FIX in order to be accurate required to be trained on appropriate patient data.•FIX in contrast to AROMA additionally increased RSNs identifiability. RestingHowever, overcoming the contribution of noise in the acquisition of rs-fMRI data is important in limiting false observations and reliably estimating functional connectivity in Resting State Networks (RSN) . IndepenThere have been a variety of attempts to automate the classification of independent components (ICs) . Some avThe limitations of either approach to noise removal are potentially more apparent when applied in acute clinical populations, where multiple sources of noise may be simultaneously more evident and heterogeneous than in cohorts that are either healthy, or have a chronic disease process such as dementia. An acute stroke population is a good example of this; up to 35% of stroke patients present with aphasia , which mLimiting noise removal to a single aspect, such as motion, may not clean the data enough to enable meaningful analysis. The classification accuracy of a complex approach could diminish if the characteristics of the data to analyse deviate substantially from the training data, particularly in the presence of different scanners or acquisition protocols .In addition, grouping acute stroke patients for analysis will inevitably add further variability due to differing clinical presentation, lesion localization, haemodynamic parameters, and vasoactive medications all of which will impact the interpretation of signal changes. These wide and inconsistent potential sources of variance might undermine the clinical applicability of a complex approach to IC classification.The purpose of this study was to explore how these limitations are addressed in using ICA denoising approaches applied to a dataset obtained from a cohort of acute stroke patients. The performance of a focused classifier approach (ICA-AROMA) and complex classifier (FIX) were compared using metrics determining RSN identification, RSN reproducibility, changes in the BOLD variance and differences in the estimation of functional connectivity.22.1Consecutive patients presenting with a clinical lacunar stroke syndrome within 24 h of symptom onset (using the last seen well principle) regardless of age or stroke severity were recruited into a prospective observational cohort study following informed consent or agreement from a representative according to protocols approved by UK National Research Ethics Service committees (ref: 12/SC/0292 and 13/SC/0362). Exclusion criteria included the presence of a contraindication for MRI. All clinical decisions were made prior to enrolment in order not to introduce any delay to best clinical care.Data were acquired using a Siemens 3T Verio scanner  with a 32-channel head coil. Whenever possible, each subject was scanned at presentation, 24 h, a week and a month later. At each time point, a resting state T2*-weighted scan  and a T1-weighted MP-RAGE  were acquired.2.2Prior to the use of either ICA-AROMA or FIX, rs-fMRI data were pre-processed using EPI distortion correction, rigid-body registration, brain extraction, Gaussian smoothing with a 5 mm FWHM kernel. Each 4D pre-processed dataset entered single-subject spatial-ICA decomposition with automatic dimensionality estimation using Multivariate Exploratory Linear Optimised Decomposition of Independent Components (MELODIC) . High-pa2.3Independent components were classified into signal or noise both manually and using ICA-AROMA and FIX.2.3.1www.fmrib.ox.ac.uk/fsl), a visualization software specifically written for FIX to display each IC's spatial map, temporal power spectrum, and time course (default starting threshold used 2). Two independent raters (DC and RL) manually classified the ICs following a standardized procedure .Generic training, using the standard training-weight file supplied with FIX , created•Patient (study-specific) training using the hand labels obtained from manually classifying the fMRI data acquired from the study patients.Automatic classification using FIX was performed using 2 different training procedures:ICs that where classified as noise were subsequently regressed out from the rs-fMRI data using the non-aggressive option, ensuring that only the unique artefact-related variance was removed .2.3.3ICA-AROMA was applied after spatial smoothing and before high-pass temporal filtering. ICs that were classified as motion-related were regressed out of the data following the non-aggressive option .2.4FIX classifier performance was summarised by the ability to identify signal and noise components when compared to the agreed manual classification. Two measures were calculated: true positive ratio  and true negative ratio .FIX performance following generic and patient training on the patient data, was tested using a leave-one-subject-out (LOSO) approach across sets of ICA output components to avoid overfitting. If the training data consists of n datasets, each fold of the cross validation uses n-1 datasets for training, and tests the learned decision boundary of the left-out dataset. In the case of having multiple runs of data from each patient, it was considered safest to leave out all runs for a given patient and train on all datasets of all other patient in order to generalise LOSO accuracy results when applied to future patients.A threshold is applied to FIX output to determine the binary classification of any given component. Changing the threshold shifts the balance between TPR and TNR. Several thresholds were evaluated in order to show how to find the ideal balance between prioritizing TNR against TPR . The optAn overlap analysis was performed  to explore a possible link between the ischemic lesion localization and mislabelled components.2.5Z-scores of the fit that may be used later in group-level analyses. The distribution of the Z-scores across voxels was assessed for every map obtained. Representative histograms are included in the Supplementary Material .After pre-processing, the functional scans were registered to the MNI152 standard space  via a transformation matrix obtained from aligning the subject's functional scan to the anatomical scan, and the anatomical scan to the MNI152 template (12 DOF). Functional connectivity analysis was run both on corrected data and on original  data using a template-based dual regression  to allowwww.fmrib.ox.ac.uk/fsl) were used.In all stages, tools from the Functional Magnetic Resonance Imaging of the Brain (FMRIB) Software Library  associated with the different strategies was evaluated to investigate its potential impact on statistical power and between group bias . Loss of2.9map, was calculated (map was defined as:t-test) to determine in which regions %ΔSTD was significantly  higher than 35%, 45%, 55%, 65%. ΔSTD maps were also thresholded (25%), binarised and averaged to generate a probability map that would highlight in which areas the rs-fMRI variance was more frequently reduced across subjects.To test the effect of correction on BOLD variance, a voxel-wise variable, %ΔSTDlculated  for ever2.10The effect of ICA-AROMA and FIX on resting state connectivity was assessed by measuring the percentage of absolute change in the magnitude of Z-scores for each NOI and for each Z-score map . This pep < 0.05). A paired sample t-test was used to compare differences between FIX and ICA-AROMA .To determine if there were differences in the effect of using ICA-AROMA or FIX across NOIs, a one-way ANOVA was used with Bonferroni correction for multiple comparisons  in functional connectivity at a group level were generated for the 10 “well defined” networks ) were enrolled consecutively. Onset to first scan time ranged between 2 and 19 h. Patient factors are summarised in 3.2The average number of single-subject ICs estimated by MELODIC was 53.7 (range 35–68) per scan. The average number of ICs manually classified as signal was 8.2 (range 5–13). The inter-rater agreement ratio was 94.7% (and 94.5% after a month), whilst intra-rater agreement ratio was 95.4% for the first rater and 97.2% for the second rater.Mean and median values of TPR and TNR using different thresholds are reported in 3.3p = 0.97). Patient-trained FIX showed a significant increase in RSN identifiability , though no significant difference was found between ICA approaches  after applying ICA-AROMA and patient-trained FIX in the 10 “well defined” networks are shown in The spatial maps representing significant group-level changes in functional connectivity (3.8Twenty grayplots (selected at random from the 60 possible) are presented in the Supplementary Material . Green lines and blue lines indicate voxels in the white and gray matter, respectively. Motion artefacts  appear to be attenuated, but not completely removed by the use of either ICA-AROMA or patient-trained FIX.4ICA based denoising approaches were successfully applied to rs-fMRI data obtained from acute stroke patients resulting in improved RSN reproducibility, localized reduction in BOLD variance consistent with noise removal and significant changes in functional connectivity. Compared to ICA-AROMA, patient-trained FIX resulted in higher RSN identifiability and wider changes both in the BOLD variance and in functional connectivity.The use of all ICA strategies led to a significant increase in RSNs reproducibility reflecting the reduction of the group level variability due to noise. Following the use of ICA-AROMA, changes in the BOLD signal variance were both more frequent and more prominent in proximity of the brain edges, consistent with the intended removal of motion-related artefact. The changes in BOLD signal variance after the use of patient-trained FIX were more extensive, also involving regions in proximity of large vessels  suggesting the ability to capture and remove physiological noise (vascular and CSF pulsation artefacts).The removal of noise may come at a cost. Decreased tDoF might undermine the reliability of subject-level functional connectivity estimates and introduce a bias in group-level analyses due to between-subject tDoF-variability . This stWhen it came to the assessment of the preservation of the signal of interest, this study demonstrated findings consistent with previous results following the use of ICA-AROMA , confirmThe use of generic-trained FIX in this acute stroke population proved to be a non-viable choice due to significant issues with misclassified and discarded signal components. The FIX generic training weight dataset was developed on an identical scanner and using identical acquisition protocols to those used in this study. These issues with FIX accuracy, therefore, suggest the presence of disease specific effects altering both noise and signal components and that these effects are at least as important as those introduced by either scanner or acquisition protocol.This study demonstrates the necessity of the appropriate training of FIX. Appropriate training of FIX in this specific cohort needs to take account of the dynamics of both noise and signal components over the first month following stroke. This will include the transition from the effects of injury in the first scans to the effects of recovery and plasticity seen in the later scans. The patient-training dataset was developed using data from a broad range of patients from all time points across the first month following acute stroke. Its use resulted in an increase in FIX accuracy, not least through the increase in RSN identifiability reflecting higher signal to noise levels, even in a population with such wide and heterogeneous sources of variance as acute stroke patients.http://www.fmrib.ox.ac.uk/analysis/FIX-training/) for testing in separate populations of acute stroke patients. If healthy controls are to be included in an analysis, a different training dataset will likely be required  and a complex (patient-trained FIX) classifier approach result in significant noise removal from rs-fMRI data acquired from a population of acute stroke patients. The success of ICA-AROMA demonstrates that by concentrating on a restricted number of components related to a very common cause of noise, namely motion, the full automation of an ICA-based approach can deliver meaningful data for analysis even in population with such heterogeneous sources of noise. However, the time invested to train FIX with appropriate patient data encompassing the multiple sources of disease-specific noise (in addition to those that are scanner- and acquisition-related) proved valuable, particularly in improving the signal-to-noise ratio. Depending on the application, both approaches clearly have a role in future stroke studies: ICA-AROMA might be preferred in the setting of noise removal from rs-fMRI data in a multi-centre study where individual site patient numbers might be low or the expertise to develop the training dataset might be limited (both impeding the development of a site-specific training dataset), and the scanners and acquisition protocols might be varied across sites; whereas, patient-trained FIX might be have a role in individual proof of concept studies where the resulting improvement in signal-to-noise ratio means that committing resources to training FIX appropriately are important.


He ultimately recovered and was discharged without complications.A 35-year-old, otherwise-healthy male presented to a military emergency department complaining of persistent cough. He had been treated for community-acquired pneumonia three weeks prior to this presentation with a five-day course of azithromycin. His vital signs were stable with no fever, hypoxemia or respiratory distress. His physical exam was unremarkable with no focal lung findings. Chest radiograph  was perfPulmonary POCUS can be used at the bedside to diagnose and describe pleural effusions. The exam is performed using a low-frequency probe placed in the posterior axillary line in a longitudinal view. Fluid collections will be visible directly above the diaphragm. Transudates will appear anechoic and simple on ultrasound. A more complex appearance with complex septations or heterogenous appearance indicates the presence of an exudate.What do we already know about this clinical entity?An empyema is a complication of pneumonia where the pleural cavity is filled with a purulent effusion.What is the major impact of the image(s)?Pulmonary point-of-care ultrasound can improve the early diagnosis and differentiation of pleural effusions when compared to chest radiography.How might this improve emergency medicine practice?Incorporating point-of-care ultrasound early in the management of patients with abnormal lung findings may improve care by improving diagnostic accuracy.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.VideoThoracic point-of-care ultrasound using 4-megahertz curvilinear probe, demonstrating an empyema in a coronal view (arrow).


The prognosis for recovery of motor function in motor complete spinal cord injured (SCI) individuals is poor. Our research team has demonstrated that lumbosacral spinal cord epidural stimulation (scES) and activity-based training can progressively promote the recovery of volitional leg movements and standing in individuals with chronic clinically complete SCI. However, scES was required to perform these motor tasks. Herein, we show the progressive recovery of voluntary leg movement and standing without scES in an individual with chronic, motor complete SCI throughout 3.7 years of activity-based interventions utilizing scES configurations customized for the different motor tasks that were specifically trained . In particular, this report details the ongoing neural adaptations that allowed a functional progression from no volitional muscle activation to a refined, task-specific activation pattern and movement generation during volitional attempts without scES. Similarly, we observed the re-emergence of muscle activation patterns sufficient for standing with independent knee and hip extension. These findings highlight the recovery potential of the human nervous system after chronic clinically motor complete SCI. Activity-based training  is not sufficient to promote significant restoration of motor function in this population6. However, it can induce positive neurophysiological adaptations in the spinal circuitry and, to some extent, modulate the motor pattern generated during assisted standing and stepping. Studies in animal models with complete SCI have demonstrated the potential of activity-based training to promote the recovery of lower limb motor function for standing and stepping10. This recovery is substantially improved with the combination of spinal cord epidural stimulation (scES) and also pharmacological intervention15. These findings led to the hypothesis that humans with motor complete SCI may be able to recover independent standing or stepping performing locomotor training with scES. We tested this hypothesis in four chronic clinically motor complete SCI individuals, and showed that stand training performed with individual-specific configurations optimized for standing (Stand-scES) progressively promoted the recovery of full body weight bearing standing with self-assistance for balance18. It was also observed that step training with scES optimized for stepping (Step-scES) performed subsequently to stand training impaired standing performance in three of these four participants, underscoring the relevance and task-specific effects of the trained motor task. In addition, delivering sub-motor threshold levels of scES customized for voluntary movement (Vol-scES) enabled these individuals to perform relatively fine volitional lower limb movements from the supine position in response to conceptual, auditory and visual input19. The presence of Stand-scES was always required to stand in all four individuals, and Vol-scES was always required to elicity voluntary leg movements in three of these research participants. This report describes the unexpected recovery of lower limb voluntary motor control and independent standing without scES in one of these chronic, motor complete SCI individuals that progressed throughout 44 months of activity-based training with scES.The prognosis for recovery of lower limb motor function in individuals with chronic clinically motor complete spinal cord injury (SCI) is poor18 was enrolled to perform additional activity-based training with scES at home and in the laboratory following the completion of the initial study. Research participant B13, a 32-year old male, was implanted with a spinal cord epidural stimulation unit 4.2 years after SCI caused by a motorcycle accident. Prior to stimulator implant, this individual was unable to stand or walk independently or voluntarily move his legs despite standard-of-care rehabilitation and locomotor training received during the initial 21 months following the injury. Additional intensive locomotor training (80 sessions) performed prior to the stimulator implant did not result in functional improvements for standing, stepping or voluntary movement. Two clinicians independently performed a physical exam following the International Standards for Neurological Classification of Spinal Cord Injury21 prior to stimulator implant, and classified the individual as AIS B (pinprick and light-touch present below the lesion), with a neurological level of injury at C7. In addition, as reported in a previous publication19, no functional motor connectivity between the supraspinal and spinal centers below the level of injury was detected in this research participant. The individual signed an informed consent for electrode implantation, stimulation, activity-based training and physiological monitoring studies approved by the University of Louisville and the University of California, Los Angeles Institutional Review Boards. All research activities were performed in accordance with the guidelines and regulations of these Institutional Review Boards. The research participant and the other persons appearing in the Supplemental Videos included in this paper also gave written informed consent and granted full permission for their image to be used in publication online.One of the four research participants with chronic motor complete SCI who were recruited to investigate the effects of activity-based training with scES on the recovery of lower limb motor function16. The electrode lead was tunneled to a subcutaneous abdominal pouch where the pulse generator was implanted. Representative scES parameters utilized during activity-based training are reported in Supplemental Table The lumbosacral enlargement was electrically stimulated during activity-based training using an epidural spinal cord stimulation unit  and a 16-electrode array  that was implanted at the T11-L1 vertebral level over the spinal cord segments L1-S123. During hip flexion at experimental time points t1 to t5, a non-elastic cable was secured to the ankle and attached to a fixed frame; this cable allowed a maximum hip flexion of 30 degrees.The data reported in the present study were collected without scES at different time points, over a period of 4.1 years  was recorded with fine-wire electrodes. Lower limb joint angles were acquired using a high-speed optical motion capture system . Ground reaction forces were collected using a high-resolution pressure sensing mat  or two force platforms .EMG, kinematics and ground reaction forces data were recorded at 2000 Hz using a custom-written acquisition software . EMG activity of right (R) and left (L) gluteus maximus (GL), medial hamstring (MH), rectus femoris (RF), vastus lateralis (VL), tibialis anterior (TA), medial gastrocnemius (MG), soleus (SOL), sternocleidomastoid (SCM) and intercostal  muscles were recorded with bipolar surface electrodes with fixed inter- electrode distanceData analysis of volitional attempts was performed on a 3-second time window, the onset of which coincided with the activation of the primary agonist muscle . If the primary agonist muscle was not active throughout the whole attempt, the time window onset corresponded to the beginning of the volitional attempt as estimated by the activation of intercostal or sternocleidomastoid muscles.24 to obtain quantitative information about the coordination pattern between representative agonist and antagonist muscles during volitional movement attempts. Each data point in the JPD represents the amplitude relationship of the EMG signals from the two muscles at a given time point. Ten percent of the full scale value was then set as a threshold in order to define the four areas  represented in Fig. The amplitude of EMG activity during volitional attempts and standing was quantified by root mean square (RMS) and normalized (divided) by background (resting) RMS EMG amplitude that was recorded prior to the volitional attempts or during sitting, respectively. Hence, a normalized EMG amplitude value equal to 1 indicates that the EMG amplitude detected during the examined motor task  was equal to the background (resting) EMG amplitude. Joint probability density distributions (JPD) was calculated as reported by Hutchison and colleagues25). This training was intented to achieve any positive adaptations induced by activity-based rehabilitation before the beginning of training with scES.Prior to epidural stimulator implant  on a treadmill  in a home-based setting and once a week in the laboratory for 9.5 months , or motor and sensory complete (AIS A) lesions29. However, these descending inputs were not sufficient to activate motor pools in the absence of scES.We observed substantial recovery of volitional lower limb motor control and independent standing ability without using scES in an individual with chronic, motor complete SCI thoughout long-term activity-based training with task-specific scES parameters. At the beginning of the study, attempts to perform hip flexion or knee extension without scES did not induce any volitional movement or EMG activity from the lower limb muscles Figs  and 4. H33. Co-activation of agonist, antagonist and distant muscles as well as multi-joint movements of the lower limb are also often observed in clinically motor incomplete SCI individuals when attempting to perform single-joint movements35. The inability of these individuals to perform isolated muscle activation leading to single-joint movements may suggest that remaining supraspinal motor input to the spinal cord was sufficient to initiate a motor pattern, but had limited capacity to selectively activate specific muscles of a single joint or to simultaneously inhibit antagonist muscles. At t8, further improvements in the volitional motor patterns were observed. In particular, during both hip flexion and knee extension attempts, a substantial decrease in the co-activation of antagonists and distant muscles was detected  also may have been reorganized by long-term activity-based training with scES, having a role in the observed refinement of volitional control39.Interestingly, stand training with Stand-scES and voluntary movement training with Vol-scES were sufficient to modify the motor output generated during volitional attempts to move the lower limbs without spinal stimulation. In fact, the intent to perform hip flexion and knee extension at experimental time point t2 resulted in the co-activation of agonist and antagonist muscles as well as distant leg muscles, and in the ability to perform hip flexion from the supine position Figs  and 4. S40, in 5 individuals after activity-based training with transcutaneous spinal cord electrical stimulation combined with pharmacological intervention36, in three individuals after functional electrical stimulation-assisted locomotor training and continuous pelvic-lumbosacral neuromodulation41, and in a sub-group of individuals after long-term activity-based interventions enriched by virtual reality and multi-sensory feedback42. All these interventions activated the neuromuscular system below the level of injury with activity-based training. The time course of assessments presented in this study uniquely demonstrates the re-emergence of volitional motor control and its evolution over time, which seem to mirror the pattern of recovery frequently observed after incomplete SCI39. These findings suggest that when there is no ability to voluntarily generate movement below the level of injury, residual descending input to the spinal circuitry may be accessed with intense activity-based interventions to result in plasticity that can promote volitional movement recovery.Limited recovery of volitional motor control after chronic motor complete SCI were observed in one individual after a 3-year period of functional electrical stimulation bicycle therapy6. Similar findings were observed in the present study at experimental time points t1 to t5. In fact, the research participant was able to achieve and maintain the upright position only via external assistance for hips and knees extension. In addition, EMG activity recorded during assisted standing at the experimental time points t1 to t5 was negligible and comparable to that recorded in sitting ing Fig. . Similar43. In the present study, one of the most relevant improvements, the generation of motor patterns sufficient for standing independently, was observed for the first time after a 3-month training period (t5 to t6) that began 21.5 months after stimulation implant. It is worth noting that this training paradigm differed substantially from previous training in terms of: i) frequency of the training stimulus; ii) motor tasks trained in a day; and iii) volitional involvement of the research participant. In particular, during this period, two 1-hour training sessions per day were performed (1 stand training with Stand-scES and 1 step training with Step-scES) instead of the 1-hour daily training previously performed. The choice to train daily both standing and stepping rather than focusing on one motor task was based on previous findings that showed as standing ability impaired after a period of step training in three out of four motor complete SCI individuals implanted with a scES unit18. Hence, we proposed to train both standing and stepping in an alternating fashion with the hypothesis that this approach would promote improvements in both motor tasks. In addition, the research participant was strongly encouraged to volitionally contribute to the modulation of activation pattern during standing and stepping. In fact, it was previously shown that EMG pattern during assisted stepping with scES can be dramatically modulated by the intent of the clinically motor complete individual to contribute to the stepping pattern generation19, although this approach could lead to an overall impaired motor output (i.e. greater co-contraction during assisted stepping). Another difference was that the main goal of this training paradigm was no longer to complete the training session with the least amount of seated rest regardless of independence level, but rather to decrease external assistance regardless of the amount of seated rest required. Motivation and goal-oriented training were shown as important for the recovery of voluntary control of locomotion after SCI in rats, inducing growth of denovo brainstem and intraspinal relays15. Conversely, rats that were trained in a more passive and automated manner (treadmill-restricted training), which did not promote oriented cortical activity, did not show functional recovery overground or neural plasticity across the lesion. While in the present study we have not collected any data related to cortical activity, we have observed that the research participant attention during training sessions performed between t5 and t6, and between t7 and t8, was markedly focused on the trained motor task, attempting to volitionally contribute to the motor output. Therefore, it can be hypothesized that the increased volitional involvement of the research participant during stand and step training may have also contributed to the improved motor execution observed between t6 and t8, possibly promoting further intra- and supra-spinal plasticity.Activity-based training is one of the main determinants of the motor improvements observed in the present study. However, little is known about the influence of different components of activity-based training  on physiological adaptations resulting in motor function improvements after SCIIn conclusion, long-term activity-based training with scES promoted recovery of both volitional lower limb movements and standing without using scES in an individual with a chronic, motor complete SCI. In particular, this is the first report detailing the ongoing neural adaptations that allowed a functional progression from no volitional muscle activation to a refined, task-specific activation pattern during volitional attempts, as well as to the generation of activation patterns sufficient for standing. Novel characteristics of the training paradigm that were implemented after experimental time point t5  were conceivably important for the functional improvements herein described. These findings highlight the remarkable recovery potential of the human nervous system after chronic clinically motor complete SCI in response to activity-based training with scES, and have clear implications for the rehabilitation community. Future studies should attempt to better understand the influence of different activity-based training components  on the physiological adaptations resulting in motor function improvements after severe SCI.Supplementary InformationSupplemental video S2Supplemental video S3


Membrane depolarization significantly induced axonal branch-extension in cultured cerebellar granule neurons (CGNs). Local application of nifedipine using a polydimethylsiloxane (PDMS)-based microfluidic device demonstrated that Ca2+ entry from the axonal region via L-type voltage-dependent calcium channels (L-VDCC) is required for branch extension. Furthermore, we developed a method for locally controlling protein levels by combining genetic techniques and use of a microfluidic culture system. A vector for enhanced green fluorescent protein (EGFP) fused to a destabilizing domain derived from E. coli dihydrofolate reductase (ecDHFR) is introduced in neurons by electroporation. By local application of the DHFR ligand, trimethoprim (TMP) using a microfluidic device, we were able to manipulate differentially the level of fusion protein between axons and somatodendrites. The present study revealed the effectiveness of microfluidic devices to address fundamental biological issues at subcellular levels, and the possibility of their development in combination with molecular techniques.The establishment and maintenance of axonal patterning is crucial for neuronal function. To identify the molecular systems that operate locally to control axonal structure, it is important to manipulate molecular functions in restricted subcellular areas for a long period of time. Microfluidic devices can be powerful tools for such purposes. In this study, we demonstrate the application of a microfluidic device to clarify the function of local Ca


Sedimentibacter sp. and gammaproteobacteria). These differences in the microbiome may be responsible for the two-fold increase in power production achieved by the MPPT-MFC compared to the Ref-MFC.Microbial fuel cells (MFCs) can be designed to combine water treatment with concomitant electricity production. Animal manure treatment has been poorly explored using MFCs, and its implementation at full-scale primarily relies on the bacterial distribution and activity within the treatment cell. This study reports the bacterial community changes at four positions within the anode of two almost identically operated MFCs fed swine manure. Changes in the microbiome structure are described according to the MFC fluid dynamics and the application of a maximum power point tracking system (MPPT) compared to a fixed resistance system (Ref-MFC). Both external resistance and cell hydrodynamics are thought to heavily influence MFC performance. The microbiome was characterised both quantitatively (qPCR) and qualitatively (454-pyrosequencing) by targeting bacterial 16S rRNA genes. The diversity of the microbial community in the MFC biofilm was reduced and differed from the influent swine manure. The adopted electric condition (MPPT vs fixed resistance) was more relevant than the fluid dynamics in shaping the MFC microbiome. MPPT control positively affected bacterial abundance and promoted the selection of putatively exoelectrogenic bacteria in the MFC core microbiome ( Continuous and unsustainable animal production causes an accumulation of undesirable products such as swine manure, which has a complex organic matter and nitrogen (primarily ammonium) content that contributes to environmental pollution. Annually, 1.4 billion tonnes of swine manure are generated in the European Union, where the main contributor country is France ,3. Microin situ swine manure treatment. MFCs will become a recognisable alternative technology for green energy recovery from wastewater treatment in the near future. Scientific reports on MFCs include the evaluation of their nutrient removal capacities . Com. Com36].(2.7 mW) . Moreove studies .In this study, different external resistance control strategies were applied to two replicate MFCs fed swine manure. The effect on the development of the exoelectrogenic bacterial community was evaluated to optimise the internal MFC bioprocesses. A MPPT control strategy (based on dynamic resistance) was applied to one of the MFCs and compared with a Ref-MFC operated at fixed resistance. Microbial groups developing in the anode chamber were identified. The comparative analysis of the MFC core microbiomes allowed the identification of bacteria that were potentially responsible for the power generation. Moreover, the internal distribution of the bacterial abundance was analysed and related to the reactor fluid dynamics.et al. (2014). Briefly, each MFC consisted of a methacrylate, rectangular reactor with an anode and cathode placed on opposite sides of an Anionic Exchange Membrane . Each anode and cathode chamber contained approximately 400 mL of liquid volume . The cathode was fed an oxygen-saturated inorganic solution (see Supplementary methods). The temperature was kept constant at 21 ± 1°C.Two replicate, dual-chamber MFCs were constructed and operated to remove organic matter from swine manure with bioelectricity production. Details of the MFC design and operation are described in the study of Molognoni d volume  39]. Sw. Swet alThe two MFCs operated under the same hydraulic conditions and differed only in the electrical load application . The MPPet al. 68],[68],Prot,[68],Pro glucose 19], rev, revProtc matter .Turicibacter-related species may be implicated in heterotrophic degradation of organic matter, probably through a fermentation process. However, additional molecular analyses, including shotgun metagenome and metatranscriptome sequencing, will be necessary to identify putative exoelectrogenic bacteria in MFCs based on functional capacity and activity and confirm the previous hypothesis.The effect of the MPPT control on the bacterial community structure was analysed by comparing the microbiome core communities and related to the increment of current density. The core community of MPPT-MFC contained three different OTUs that appeared at significantly higher relative abundances compared to Ref-MFC. Interestingly, the only OTU found at higher abundances in Ref-MFC was OTU 0, thus questioning its implication in exoelectrogenesis since significantly lower CE was found for Ref-MFC. More likely, Sedimentibacter spp. (OTU 10), Pseudomonas sp. (OTU 12) and an uncultured Oxalobacteraceae (OTU 23) were significantly enriched in the MPPT-MFC. Based on the analysis of 16S rRNA gene similarities, Sedimentibacter related species were identified in the core community of MFC systems with high power generation capabilities together with Geobacter, Aminiphilus, Acetoanaerobium, and Spirochaeta [Pseudomonas species [rochaeta . Althoug species .vs fixed resistance) was more relevant than the fluid dynamics in shaping the MFC microbiome. The MFC core community was primarily composed of the fermentative Turicibacter genus. The MPPT control was able to select specific OTUs potentially harbouring higher exoelectrogenic capacities compared to the fixed resistance system. Sedimentibacter and gammaproteobacteria were among the most abundant phylotypes that being a member of the core community were enriched in the MPPT-MFC compared to Ref-MFC. Hence, it is likely that these organisms may be related to the extra electricity production in the MPPT-MFC. The optimization of the MFC systems together with the comprehension of the bacterial communities responsible for the internal processes will enable the implementation of MFC technology for in situ swine manure treatment. For this reason, future studies could focus on the physical relationship of the dominant taxa with the electrode  and the identification of active members of the MFC-associated community (shotgun metatranscriptome sequencing).The MFC microbial community diversity and abundance were studied in relation to the external resistance control and fluid dynamics. The bacterial community from the studied MFCs was more efficient at treating the complex organic matter than the community reported in previous studies. The application of a MPPT electric control in MPPT-MFC resulted in a 5-fold higher bacterial abundance compared to the Ref-MFC and doubled energy production and CE. The adopted electric condition Click here for additional data file.S2 Fign = 4 for Ref-MFC and MPPT-MFC, and n = 2 for Swine Manure (SM).(DOC)Click here for additional data file.S3 FigAll pair-wise combinations of data-points have been organized as Inter-group or Intra-group comparisons and shown as individual points using different colours.(DOC)Click here for additional data file.S1 File(DOC)Click here for additional data file.S1 TableThe values are presented as average ± standard deviation (n = 7). n.d. not detected. * Acetic acid and Propionic Acid were the only VFA identified and detected above LOD.(DOC)Click here for additional data file.S2 TableCore community members were limited to those OTU that were found in at least three of the four samples in the MFC. Differences were assayed using a t-test. FDR  correction of p_value. n.d. not detected. NS- not significant .(DOC)Click here for additional data file.


Melanoma is one of the most immunogenic tumors and its relationship with host immune system is currently under investigation. Many immunomodulatory mechanisms, favoring melanomagenesis and progression, have been described to interfere with the disablement of melanoma recognition and attack by immune cells resulting in immune resistance and immunosuppression. This knowledge produced therapeutic advantages, such as immunotherapy, aiming to overcome the immune evasion.Here, we review the current advances in cancer immunoediting and focus on melanoma immunology, which involves a dynamic interplay between melanoma and immune system, as well as on effects of “targeted therapies” on tumor microenvironment for combination strategies. Cutaneous melanoma (CM) is the most common skin cancer with an incidence that is rapidly increased in the past decade . It is eelimination of tumor cells based on immunosurveillance, equilibrium between tumor and immune cells and escape or immune evasion. The elimination is based on sequential events leading to the anti-melanoma cytotoxicity by natural killer (NK) and dendritic cells (DCs), T-cells, B-cells either within the tumor microenvironment or in peripheral tissues [Rag)-1 or Rag-2 fail to rearrange the lymphocyte antigen receptors, thus resulting in defective production of mature peripheral B cells as well as NK T cells (NKT) or αβ and γδ T cells. Moreover, Rag-2−/− mice subcutaneously injected with the chemical carcinogen 3′-methylcholanthrene (MCA) showed a high incidence of sarcomas with respect to wild-type controls [−/− and TCR δ−/− mice. In addition, the primary role of αβ and γδ T cell subsets has been also demonstrated in promoting functional antitumor immune response [During the melanomagenesis , both ce tissues . The equ tissues . The equcontrols , while fresponse  and IFN-The functional exhaustion of the immune system depends on the persistent antigen exposition by melanoma cells and induces the hyper activation of inhibitory checkpoints on immune cells as result of a negative feedback for the cytotoxic T-cells , 9. SimiBased on the knowledge of the melanoma biology, new therapeutic strategies including CTLA-4, PD-1 and PD-L1/2 blockers, aimed at restraining the molecular interplay between tumor cells and effector immune cells, have been developed and results from extended clinical trials describe a revolutionary improvement in the management of melanoma patients –18.Here, we revisit both cellular and molecular events that balance immunosurveillance and editing in melanoma biology as major mechanisms involved in tumor progression and a brief description of the innovative therapeutic strategies against this tumor.Immunogenicity of a tumor is the capacity to induce adaptive immune responses that can prevent its growth and melanoma cells are considered highly immunogenic or capable of activating or modulating the adaptive immune response resulting in the balance between survival and proliferation . HoweverGenerally, the immunogenicity has been considered for long time the boundary to discriminate self from non-self. Therefore, the immunogenicity requires that melanoma cells express adequate levels of antigens capable to elicit immune activation instead of immune tolerance. This, however, depends not only on the antigenicity of cancer cells but also on functional interactions among immune cells as well as on immunomodulatory factors released by the tumor cells .The reasons underlying the immunogenicity  of melanThe molecular mechanisms that regulate the immune response in melanoma and its spontaneous regression sporadically observed in the clinical setting are unclear and are partially explained on the number of somatic mutations of coding exons and/or the splice junctions that recur in melanoma cells as well as on the high mutational load and the mutagenic signature induced by ultraviolet light . AdditioThe most relevant antigens expressed by melanoma cells include: i) differentiation antigens or tumor-associated antigens (TAAs) expressed by both normal and malignant melanocytes; ii) cancer/testis antigens  that areTherefore, the neo-antigens are considered ideal targets for immunotherapy and their detection could be a good biomarker to predict the efficiency of immune checkpoint inhibitors including both anti-PD-1 (programmed death-1) and anti-CTLA4 monoclonal antibodies (MoAbs) , 29. In A number of studies proved the relationship between defective immune system activity and melanoma cell proliferation, while others demonstrated that the variability of the antigenic repertoire is a critical factor for the immunosurveillance and melanoma progression , 32.+ cells, or in patients whose treatment response is directly correlated to the density of TILs nearby the tumor cells [An efficient anti-melanoma immune response requires a fast and not-specific phase that activates the innate immunity before a specific adaptive stimulation of the immune system. Both phases induce apoptosis of melanoma cells through T-cell-mediated cytotoxicity and the efficiency of both T-cells and signals driven by TCR are thus central to counterbalance the melanoma cell growth although the quality of cross-priming, antigen presentation and immune cell recruitment within the tumor bed remain critical factors during the melanoma progression . This haor cells –37.+ and CD8+ T-cells (Teffs) primed against melanoma cells through the endogenous production of interferon-gamma (IFN-γ), a direct cytotoxicity tumor resulting from MHC-TCR interaction or the antigen-dependent activity of T-cells during the adaptive immune response [The tumor immunity cycle is a process based on: a) a not-specific early phase driving the innate immunity that is mediated by macrophages, granulocytes, DCs and NK cells; b) late functional phase of effector CD4response .a) NK cells and DCs: NK cells participate to the anti-melanoma immunity. In particular, they recognize and attack melanoma cells expressing low MHC class-I molecules with higher efficiency then T-cells [+ cells [ T-cells  and prom T-cells . Moreove T-cells . In this[+ cells . It has [+ cells , 44.+ immunity and the modulation of T helper activity [Mature DCs balance the efficiency of immune response and the ability of T-cells to orchestrate a cytotoxic effect. They physiologically circulate in peripheral blood and migrate to lymph nodes where they encounter naïve or memory T cells . Mature activity .b) Teff cells: the efficiency of both CD4+ and CD8+ cells for the modulation of the adaptive immune response mostly depends on the specificity of the TCR signalling. Teffs play a pivotal role during the cell-mediated immunity through TCR-MHC interactions that is powered by IFN-γ and TNF-α. The primary role of adaptive immunity in melanoma is also addressed by the brisk T-cell infiltration that is considered a positive prognostic issue [+ Teff cells commonly do not capture melanoma antigens from cells lacking MHC class-II molecules, although a number of studies proved that the majority of melanoma cells are restored in class-II expression by high levels of IFN-γ. The expansion of T lymphocytes activated against melanoma cells through the clonally distributed TCR, leads to the formation of elevated numbers of mRNA encoding the α and β chains of TCR. In particular, infiltrating T cells from murine melanoma models bear clonally expanded TCR transcripts whose activity is of great effort for the efficiency of the anti-melanoma immunity. In addition, several studies demonstrate that melanoma is characterized by a high number of clonally expanded T cells. However, the ‘selectivity’ of TILs is the consequence of the balance between clonality and specificity [ic issue . Moreovecificity .The general findings are that antitumor response may involve a variety of clonal TCRs that, notwithstanding a similar structure, may recognize the same HLA/peptide complex. Therefore, the relation between specificity and clonality has been clearly demonstrated proving that the clonally expanded cells within the tumor microenvironment are tumor-specific.Finally, the attack of T-cells against melanoma cells results in their apoptosis by the release of perforin and granzymes followed by new antigen diffusion within the tumor bed. These sequential events allow the immunity cycle to start again and self-sustaining .In conclusion, the results of this T-cell recruitment and activity within the tumor microenvironment predominantly include the induction of apoptosis in melanoma cells followed by the release of new antigens which once again allow to restart and self-maintaining of the anti-tumor immune cycle .‘immune editing’ that is mostly based on a complex machinery of intra- and extracellular signals. Immune editing is a dynamic process putatively involved in the colonization of distant tissues by melanoma cells and includes the ‘immune escape’ phase. It is driven by the chronic stimulation of the immune system and by strategies of malignant cells to counteract the immune-mediated antigenic recognition. Besides the progressive exhaustion of immune system to counterattack highly proliferating malignant cells, the escape also depends on the defective immune recognition as well as on the increased resistance to apoptosis of melanoma cells, or the development of an immunosuppressive microenvironment [The escape of melanoma cells from the immune system control is regulated by the ironment , 58.+ T-cells to recognize the processed target antigens of the tumor cells [The defective immune recognition is the first event useful to the melanoma cells for the immune escape and is due to inefficient antigen processing machinery  that proor cells . Moreoveor cells  is also or cells , 63.+ and CD8+ lymphocytes, and NK cells. Among these cytokines, IDO inhibits the effector T-cells by depleting tryptophan, while promotes the differentiation and activation of Foxp3+ Tregs by kynurenine production [Besides an intrinsic defect of DCs in their ability to arm the immune system against melanoma cells and the negative influence exerted by soluble factors produced by malignant cells, other populations including MDSCs , 65 and oduction .Another functional mechanism potentially affecting the anti-melanoma T-cell activity includes the defective expression of immune checkpoint receptors as well as their intrinsic binding capability by relative ligands . The T-cBRAF are critical drivers of the melanoma proliferation in approximately 50-60% of cutaneous melanomas [NRAS recur in 10-20% of patients [The RAS/RAF/MEK/ERK cascade is the major deregulated pathway in malignant melanocytes. Mutations of elanomas  whereas patients  and are patients –82 is acpatients .+ T-cells nearby tumor cells and increases the exposition of TAAs synthesized by melanoma cells. Moreover, after exposure to BRAF inhibitors, a relationship between the number of infiltrating CD8+ cells and tumor burden following BRAF blockade has been described and the increased intratumoral CD8+ lymphocytes infiltration is apparently correlated with the reduction of the tumor size and enlarged necrosis in biopsies [Besides a specific anti-melanoma effect, however, targeted agents may potentially restore the immune system activity and early studies proved that inhibition of BRAF cascade raises the number of CD8biopsies . Furtherbiopsies . On the biopsies  that arebiopsies  a relatibiopsies . Other pbiopsies  and the biopsies .in vitro and in vivo of NK cells [V600E melanoma cell lines in terms of maturation, antigen capture, cross-priming and production of IL-12 and TNF-α [The innate component of the immune system is also affected by targeted therapy and particularly the BRAF inhibitor PLX4720 increases the phosphorylation of ERK1/2, the CD69 levels, the IFN-γ secretion as well as either the proliferation or the cytotoxic activity NK cells , 92. Thend TNF-α , 93, 94.V600 mutated melanoma although the first clinical trial combining anti-CTLA4 and BRAF inhibitor resulted in highly severe hepatotoxicity [These observations provide pre-clinical evidence supporting the use of combination MAPK inhibitors and immunotherapy in BRAFtoxicity . Currentimmunosurveillance and the immune escape. The understanding of these mechanisms has been crucial for the improvement of the treatment and for the establishment of new available immunotherapy strategies. Indeed, in the last few years, the immunotherapy showed the ability to obtain clinical and relevant benefit associated with durable responses. Besides the treatment of selected immunogenic tumors, the immunotherapy is currently considered an alternative strategy in other metastatic cancers, including urothelial, kidney, colorectal, head and neck as well as non-small cell lung cancers showing objective responses [The concept that the immune system plays a critical role in controlling the tumor progression of melanoma is well established. Studies of the tumor microenvironment and the “immune contexture” , 97, undesponses , 98–100.The next objective in the treatment of this cancer includes the full recruitment of the immune system activity by targeting co-inhibitory and co-stimulatory molecules in sequence or in combination with the targeted therapies such as the BRAF/MEK inhibitors, the approaches to improve the function of innate immune response, the cytokines, the IDO inhibition, the adoptive cell transfer and T-cell engineering, the therapeutic vaccines in combination with ongoing therapeutic approaches .The research of soluble and local immunologic biomarkers or the corresponding molecular profile of melanoma could also provide the valuable prognostic and predictive knowledge for the best therapeutic decision and identify novel targets for immunotherapy. However, melanoma remains a “model cancer” to increase our understanding of the immune-oncology to be also extended to other tumor types.


Without cell lysis, the detection limit for intracellular ATP analysis was 40 tumor cells. Furthermore, chemosensitivity of four strains of tumor cells  was determined by this assay successfully. The cell penetration ability of TAT-LUC enables the assay not only to reflect drug resistance of tumor cells real-timely but also to minimize the test time, which can be a valuable aid for personalized cancer chemotherapy.The analysis of intracellular ATP can reveal the response of cells to different treatments and is important for individualized medicine. In the present study, we developed a cell penetrating peptides (CPPs) tagged luciferase (TAT-LUC) for tumor chemosensitivity assay. The activity of recombinant TAT-LUC was evaluated using ATP standard solution and tumor cells. This recombinant TAT-LUC was then used for the analysis of sensitivity index (SI) of four strains of tumor cells. The results showed that TAT-LUC could detect less than 10 nM extracellular ATP with a strong correlation between the luminescence intensity and the ATP content (R Each result is represented as mean ± SD. To detect statistical correlations, the Spearman’s rank correlation coefficient (RE. coli DH5α host cells. The recombinant plasmids were extracted and identified by restriction enzyme digestion. The resulting product corresponded to a 1600 bp band in the gel (The constructed TAT-LUC is consistent with the forecast amplification band close to the 1500 bp Marker . The TAT the gel . Sequenc the gel .E. coli BL21(DE3) and purified by Ni-NTA affinity chromatography. The expression and purification of TAT-LUC were analyzed by SDS-PAGE. Compared with the uninduced group , activity of TAT-LUC was also proportional to the ATP concentration in cell-free luciferase assay (2 = 0.994). Although TAT-LUC detected a relatively low luminescence intensity during the entire concentration range of ATP, the sensitivity of TAT-LUC was still capable of detecting 10 nM ATP. The time-course of transduction showed that the TAT-LUC was rapidly transduced into living tumor cells within a few minutes, while control LUC was unable to detect the intracellular ATP . A strong correlation was found between the intensity of luminescence signal and cell number over a wide range of cell numbers (2 = 0.997). In comparison, the luminescence intensity measured with LUC treatment was significantly lower than that of TAT-LUC, which explained TAT-LUC had the ability to transduce into cells. The comparison between TAT-LUC assay and cell lysis based luciferase assay was conducted in our pre-experiment. The classical luciferin-luciferase assay was able to detect 100 cells (2 = 0.996). Besides, cell lysis based luciferase assay showed that LUC detected a relatively low luminescence intensity during the entire concentration range of cell number in comparison with TAT-LUC. This indicated that ATP was partially lost during the extraction of ATP by cell lysis. The results demonstrated that TAT-LUC was a reliable and sensitivity tool for the measurement of intracellular ATP in living cells.Compared with LUC  were tested. Tumor cells were incubated with different concentrations of chemodrugs for 1–2 days. After cell culture in the TAT-LUC based assay, the culture medium was replaced with the ATP-free fresh medium containing TAT-LUC. TAT-LUC was incubated for 2 min, and the luminescence signal was recorded after culture medium was replaced by D-luciferin containing fresh medium.in vitro tumor chemosensitivity testing. In order to evaluate the TAT-LUC based assay, the sensitivity index measured by TAT-LUC based assay and MTT assay were compared. The TAT-LUC based assay was able to measure the ATP released from less than 40 cells, while the MTT assay could not detect less than 500 cells/well. The sensitivity index measured by TAT-LUC based assay was generally lower than that of MTT assay, which reflects the inhibition rate of TAT-LUC based assay was higher than MTT assay , which was proven to be reliable and sensitive in comparison with MTT assay. The greatest advantage of TAT-LUC based assay is that it allows the rapid and direct measurement of the intracellular ATP content in living tumor cells without lysing. It avoids the ATP degradation and potential operational error. Therefore, single or very little tumor specimens can be used for drug sensitivity testing, greatly improving the detection speed. Furthermore, it enables real-time reflection of tumor drug resistance and accelerates the detection rate, which can be a valuable aid for personalized cancer chemotherapy.S1 FigRed represents the TAT tag.(DOCX)Click here for additional data file.


Quantifying the accuracy of remote sensing products is a timely endeavor given the rapid increase in Earth observation missions. A validation site for Sentinel-2 products was hence established in central Germany. Automatic multispectral and hyperspectral sensor systems were installed in parallel with an existing eddy covariance flux tower, providing spectral information of the vegetation present at high temporal resolution. Normalized Difference Vegetation Index (NDVI) values from ground-based hyperspectral and multispectral sensors were compared with NDVI products derived from Sentinel-2A and Moderate-resolution Imaging Spectroradiometer (MODIS). The influence of different spatial and temporal resolutions was assessed. High correlations and similar phenological patterns between in situ and satellite-based NDVI time series demonstrated the reliability of satellite-based phenological metrics. Sentinel-2-derived metrics showed better agreement with in situ measurements than MODIS-derived metrics. Dynamic filtering with the best index slope extraction algorithm was nevertheless beneficial for Sentinel-2 NDVI time series despite the availability of quality information from the atmospheric correction procedure. Quantifying the accuracy of satellite-based remote sensing products is an important task that is increasingly timely given the rising number of Earth observation missions. Different measurement strategies are pursued at established validation sites, including continuous in situ  measurements, airborne remote sensing campaigns at the landscape level, as well as the collection of independent satellite sensor data ,2. LinkiPhenology plays a central role in the estimation of vegetation-dependent processes . ResearcThe aim of this study is to validate different satellite phenology products by comparing them to multi-sensor ground measurements. This also requires a detailed description of the site’s instrumentation, as well as information on calibration, but the main focus of this study is not on the latter. Details on calibration will be described in a separate publication . In the Compact multispectral sensors such as the SKYE SKR1860 series  are comparably low-cost and fairly straightforward to set up and maintain . HyperspIn order to compute a reflectance factor, later used for the calculation of NDVI, it is necessary to measure the upwelling and downwelling radiation fluxes. This can be achieved with different setups : systemsMeasuring downwelling irradiance and upwelling radiance quasi-simultaneously with a dual-field-of-view (DFOV) setup and different FOV fore optics ,24,25 reWe collected data at the forest site ‘Hohes Holz’ (52.08 climate . The sit climate ) and ICO climate ). The ecData logging of the multispectral sensors is performed with a Campbell Scientific CR1000 data logger with a sampling frequency of 0.1 Hz. The upward facing multispectral sensor is set up with a hemispherical cosine diffuser. The downward facing sensor is used for narrow-angle measurement  and was set up with a spectrometer slit width of 10 2.5∘ see b on a boThe sensor and shutters are controlled via LabView . Shutters open and close alternately to select the signal from upward- and downward-facing fibers to measure downwelling irradiance and upwelling radiance sequentially. Integration time was set to two seconds between April and September and four seconds between October and March. Dark current is determined with closed shutters before and after each measurement. This procedure is repeated three times every ten minutes and takes 24 seconds (three times eight seconds) between April and September and 48 seconds (three times 16 seconds) between October and March. The whole setup see  is situaHyperspectral solar irradiance data were cross-calibrated once by using measurements of solar irradiance with an ASD FieldSpec 4 spectrometer  and a Spectralon panel. Calibration performance was assessed by resampling the QE65000 spectrum to the lower spectral resolution of ASD FieldSpec. High accuracy and precision were achieved when comparing the corresponding two spectra of solar irradiance  every ten minutes and 60 times within ten minutes ; we refer to this further as the measurement-cluster. For each cluster, the absolute sum of irradiance change per channel (ect NDVI . Only upic angle .(1)ReflMean reflectance factors of each measurement cluster serve the generation of NDVI data with Equation . The res®, Version 9.1) [We used spectral data from MODIS datasets MYD09GQ  and MOD0ion 9.1)  in orderNDVI (see Equation ) is calcectively .Methods for NDVI post-processing and extraction of phenological metrics were implemented in an R-package  for phenBISE is a dynamic filter algorithm for time series. Starting at the first date of the time series , BISE accepts the next data point if it has a higher value than the previous one. Lower values are accepted if there is no point in a pre-defined time period (‘sliding period’) with a higher value. Higher values are rejected if they exceed a pre-defined threshold We applied BISE on www.phenology.de [The next step is the reconstruction of daily NDVI values based on BISE-selected NDVI observations . This dNDVI products across all systems showed very similar patterns at the forest site . The temAt the start of the season around April, NDVI is increasing until it reaches a maximum in June. The NDVI slightly decreases during the vegetation period and rapidly decreases in fall around mid-September due to senescence. It reaches its minimum value in December. The signal-to-noise-ratio decreases in winter due to snow cover, low irradiance and minor vegetation cover. Sentinel-2A images were manually examined in order to identify misclassified values and excluded if corresponding shortwave pyranometer measurements showed Green-up in 2015 and the peak of the vegetation period could be clearly identified by both MODIS NDVI and ground-based datasets. The initial NDVI increase in 2016 (March) was captured by all systems almost simultaneously. Temporal mismatches between sensor systems reduced the amount of available data points for inter-sensor comparisons . NDVI pr-2A NDVI . The mag015/2016 . NDVImulrvations .Phenological metrics calculated from NDVI time series differed between the sensor systems , althouges Holz’ a–c. Minies Holz’ d–f.GU varied between 23 April (DOY = 114) and 8 May DOY = 129) in 2016 with standard deviations lower than one week. GU calculated from es Holz’ g–i. Mini9 in 2016We validated different satellite phenology products by comparing them to multi-sensor ground measurements. The approach relates to Land Product Validation Stage 1 of CEOS (Committee on Earth Observation Satellites) . This reThe high correlation of NDVI time series between sensors suggests that the multispectral sensor system is sufficient for phenological pattern analysis. The additional use of a hyperspectral system provides, however, information beneficial for the validation of other remote sensing products  such as Differences in NDVI products are either introduced from sensor specifications , angular effects, measurement scales , calibration accuracy of satellite sensors and atmospheric correction, including cloud-detection . While sIn situ and satellite-based NDVI time series captured similar phenological patterns despite the numerous influencing factors. Correlations between the sensor systems were strong, although some deviations occurred during fall and winter between NDVI of ground sensor systems and MODIS Aqua and Terra. RMSEs between satellite and in situ data exhibited the same magnitude as found in another recent study  and as t.02×NDVI ).Depicted phenological metrics from both in situ datasets were consistent with each other and agreed with Sentinel-2A-derived metrics in 2016. Standard deviations and even the differences of green-up and senescence between the sensor systems were consistent with the natural variance of phenology. MODIS NDVI values during spring and fall were lower compared to in situ data, leading to a deviation in depicted phenological phases: green-up occurred later and senescence earlier. The later GU is contrary to the results found in . Tests wSentinel-2A images are currently available as Level 1C products. Atmospheric, cirrus and terrain corrections are necessary for further analysis. The processing tool ATCOR 2/3 produced reasonable Level 2A products. Although ATCOR 2/3 provided scene classification information supporting the elimination of outliers, misclassifications compromised the resulting NDVI time series. Three approaches can be distinguished eliminating faulty records in time series: images can be manually examined, but this is not feasible for long time series or large datasets. The usage of diffuse shortwave pyranometer measurements as selection criteria is also limited to observation sites with respective infrastructure, but these measurements are getting more and more common. Dynamic filter algorithms, such as BISE, reduce the risk of including false observations while being applicable to large datasets without the need for auxiliary site-specific measurements. Here, BISE was able to consistently remove faulty NDVI values. Daily MODIS NDVI values of 250 m lack scene classification information and respective analysis consequently requires the use of filter algorithms. BISE was able to restore the NDVI profile, although we detected mismatches with ground data during fall 2015 and winter 2015/2016. During December 2015 and January 2016, low temperatures, precipitation events and subsequently snow cover and frozen surfaces led to a strong decrease in multispectral and hyperspectral, as well as MODIS Aqua NDVI not related to vegetation. A strong increase in NDVI during the snow-melt period was detected, which is consistent with other studies observing NDVI of snow-covered vegetation ,19. The Apart from assessing the accuracy and precision of satellite products, a validation site offers the opportunity to examine data gaps or periods with low signal-to-noise-ratio introduced by bad illumination conditions and cloud or snow cover. Hence, complete ground NDVI time series are beneficial. Future studies may use a combination of DDSRR-filtering and dynamic filtering, subsequently lowering the DDSRR-threshold to obtain more data points under overcast or hazy conditions, increasing the number of usable ground measurements. The comparison of filtered satellite-based time series with in situ data provides the possibility to modify BISE parameters for satellite phenology depiction, if necessary .Here, we validated satellite phenology products for a deciduous broadleaf forest. Phenological metrics were consistent between sensor systems and agreed with the natural phenological variance also with sparse data coverage during autumn and winter time. Methods taking the effects of strongly diffuse illumination into account were already addressed in other studies ,52. In o


Apis mellifera (A. mellifera). Here we show results supporting that the supplementation of bee larvae’s diet reared in vitro with l-Arginine (precursor of NO) or ABA enhanced the immune activation of the granulocytes in response to wounding and lipopolysaccharide (LPS) injection.Many biotic and abiotic stressors impact bees’ health, acting as immunosupressors and contribute to colony losses. Thus, the importance of studying the immune response of honey bees is central to develop new strategies aiming to enhance bees’ fitness to confront the threats affecting them. If a pathogen breaches the physical and chemical barriers, honey bees can protect themselves from infection with cellular and humoral immune responses which represent a second line of defense. Through a series of correlative studies we have previously reported that abscisic acid (ABA) and nitric oxide (NO) share roles in the same immune defenses of  Apis mellifera) contribute with the pollination services which are vital to the maintenance of our ecosystem and stability of many crop yields [Honey bees  and abiotic  stressors ,3. The iHoney bees have several lines of innate immune defense against parasites and pathogens . Physical-Arginine to l-Citrulline by the enzyme NO synthase (NOS) [Nitric oxide (NO) is a highly reactive multifunctional free radical generated during the oxidation of se (NOS) . Numerouse (NOS) ,10. In iAbscisic acid (ABA) is a phytohormone that regulates fundamental physiological functions in plants  and whos(1)NO participates in granulocyte spreading .(2)Wounding induces NO production in granulocytes and augmented the total number of granulocytes in larvae hemolymph .(3)ABA supplementation in the syrup of honey bee colonies enhanced granulocyte spreading .(4)A. mellifera) in larvae hemolymph after wound challenge [ABA supplementation enhanced the number of granulocytes Varroa destructor (V. destructor) parasitism [ABA supplementation enhanced wound healing in larvae, reversing the anticoagulant effects of rasitism .(6)ABA supplementation enhanced phenoloxidase activity in adult bees .(7)ABA supplementation enhanced colony survival through winter in field approaches  and receThrough a series of correlative studies concerning honey bees, we have previously reported that:A. mellifera. The series of correlative work cited above allowed us to consider cellular spreading and production of NO as indicators of immune activation of A. mellifera granulocytes. In this work, we aimed to study if the cellular immune could be enhanced through dietary supplementation with l-Arginine (precursor of NO) or ABA in in vitro-reared honey bee larvae.As described above, we have previously reported that NO and ABA play roles in the activation of the cellular immune defense of Apis mellifera (A. mellifera ligustica–A. mellifera mellifera) larvae were transferred from brood comb to a queen rearing cup and the cups were placed into 48-well culture plates. The larval rearing plates were placed into desiccators maintained at a relative humidity of 96% (K2SO4-saturated) and 34 °C into an incubator. One day old The larvae were fed with a diet containing 50% of royal jelly, obtained from a commercial supplier, and 50% of sugar solution composed by yeast extract, D-glucose , and D-fructose . Daily diet volumes provided to the larvae and variation of the composition of the diet was done according to Aupinel et al.  , and another group of larvae were fed with the standard diet supplemented with ABA .In each independent experiment, a group of larvae were fed with the standard diet (control), another group of larvae were fed with the standard diet supplemented with l-Arg, and ABA) were immunologically challenged and evaluated as follows when they reached the first day of the fifth instar (L5).In each independent experiment, the larvae from each diet group  were selected and injected with one µL of sterile culture medium (Excell-405) alone (control of injection).Three larvae from each diet group  containing lipopolysaccharide (LPS 1 mg/mL). Lipopolysacharide is a classic inducer of NO production involved in innate immune responses in vertebrates, invertebrates, and even in plants ,23,24.l-Arg-supplemented, and ABA-supplemented) were selected as control individuals (naïve) without being injected.Three larvae from each diet treatment  with a fine needle . Twenty-four hours after the microinjections had been performed, all the injected larvae (control injected and LPS) were evaluated according to their appearance and only the larvae showing a healthy phenotype and with healed wounds were selected for further analyses. The larvae that were not selected for the experiments were discarded. Finally, two larvae were selected per group in each independent experiment gaining a total of four larvae evaluated for each of the nine experimental groups.Following the selection of the larvae, the hemolymph from the selected larvae was extracted and the immune activation (cellular spreading) and the production of nitric oxide (NO) were analyzed in each granulocyte observed within each hemocyte culture.The reason for having selected only two larvae from each diet group reduced the number of cell cultures to be observed under the microscope and this enabled an accurate evaluation of the cells. The granulocytes of each replicate were photographed and analyzed for cellular spreading and NO production (as described below). The data obtained from the four larvae of each of the nine experimental groups from the two independent experiments were pooled.l-Arginine-containing diet but that had not been injected; 62 granulocytes evaluated from larvae fed with the ABA-supplemented diet but that had not been challenged with any trauma (not-injected larvae); 71 granulocytes evaluated from blood extracted from larvae fed with the control diet that had been subjected to the injection trauma alone (injected without LPS); 76 granulocytes evaluated from the larvae fed with the l-Arginine-containing diet that had been injected with the media alone (injected without LPS); 64 granulocytes evaluated from larvae fed with the ABA-supplemented diet that had been challenged with the injection of media without LPS; 64 granulocytes evaluated from larva fed with the control diet and injected with LPS; 53 granulocytes evaluated from larvae fed with the l-Arginine-supplemented diet and that were also injected with LPS; 53 granulocytes evaluated from larvae fed with the ABA-containing diet and injected with LPS.A total of 64 granulocytes evaluated from larvae fed with the control diet but that had not been subjected to any trauma (not-injected larvae); 93 granulocytes evaluated from the larvae fed with the Hemolymph collection was performed as previously described by Negri et al. ,15,18,19A. mellifera hemocytes cultured in vitro [The endogenous synthesis of NO in living hemocytes was evidenced as in Negri et al. ,19 by mein vitro ,19.Cells that attached and spread upon stimulation with the glass surface were considered immune-related hemocytes ,18. HemoA. mellifera hemolymph were observed using inverted microscopy. Hemocytes were examined by means of differential interference contrast (DIC) or epifluorescence (excitation at 480 nm–emission at 520 nm) microscopy. The microscope used was a Nikon Eclipse Ti inverted microscope using a 60× objective.After being transferred to the 96-well plates, the mixtures containing p < 0.001) followed by the method for multiple comparisons of Dunn (p < 0.05) due the assumptions of normality and/or homogeneity of variances were violated. Statistical analyses were conducted with SigmaPlot v. 11.0 software . Data from cellular area quantification and NO production (green fluorescence levels) were analyzed using the Kruskal–Wallis method (l-Arginine and ABA dietary supplementation over granulocyte spreading and NO production were evaluated in response to LPS-injected in in vitro-reared A. mellifera larvae.The effects of As l-Arginine supplementation enhanced both granulocyte spreading and NO production when larvae had not been subjected to any challenge compared to the control group (l-Arginine-supplemented larvae showed significantly different levels of NO production compared to the control group (l-Arginine-supplemented larvae showed enhanced cellular spreading and NO production in response to LPS challenge compared to the control group (ol group A,B. In rol group A. The grol group A,B.The granulocytes from ABA-fed larvae showed stronger spreading in response to LPS treatment compared to the control group A. The prl-Arginine could work systemically as the source for NO production in honey bees. At the same time, new data reinforcing the systemic effect of ABA over the cellular immune response in A. mellifera is shown.In this work, an in vitro rearing system was used to provide with new evidence supporting the idea that dietary A. mellifera [It is important to highlight that the extraction of hemolymph is an immune challenge per se. In combination with the challenge of the hemocytes with foreign non-self surfaces like glass, this had been the immune stimulation used in previous reports in ellifera ,18. HereA. mellifera granulocytes. We focussed on evaluating those responses in granulocytes backed-up by the series of correlative work reported previously [Here, we considered the cellular spreading and the production of NO as indicators of immune activation of eviously ,18,19 suThrough the evaluation of the cellular response to injection and LPS observed in the control group, it could be speculated that the injection per se induced the immune activation of the granulocytes. This could be related with the wound generated by the needle as it had previously been reported in Negri et al. ,19.A. mellifera as it has been reported in other biologycal models [A. mellifera larvae. These results are in concordance with previous reports by Negri et al. [As it has been previously described , the chal models ,23,24. Ti et al. ,15,18,19l-Arginine is the substrate used by the enzyme nitric oxide synthase to produce NO. Here, the granulocytes extracted from the larvae that had been fed with a diet supplemented with l-Arginine showed an augmented production of NO production. At the same time, the results showed here support that both granulocyte spreading and NO production responses were enhanced by dietary l-Arginine in A. mellifera larvae. These results coincide with previous reports describing that NO participates in granulocyte activation and wound response in Drosophila melanogaster [A. mellifera [The aminoacid nogaster ,26 and Aellifera ,19. l-Arginine-fed larvae. In the present work, the response to LPS was enhanced by dietary ABA in honey bee larvae. At the same time, while the control diet group did not show an enhanced NO production in response to injection alone, the granulocytes from the ABA-supplemented larvae evidenced a significantly augmented synthesis of NO as it was also observed in A. mellifera larvae in response to wounding [The results described above are in concordance with previous reports indicating that ABA treatment enhanced cellular responses in wounding . At the wounding ,27. HoweA. mellifera immune defenses. However, more experiments should be performed to reinforce the results obtained here.All in all, the results discussed above represent new evidence supporting the roles of ABA and NO in l-Arginine could be promising nature-derived dietary supplements to enhance the fitness of honey bee colonies and could be easily coupled in the beekeepers’ practices.The search for relevant immunomodulatory tools to enhance the immune strength of honey bees represents a promising way to improve their resilience to environmental stress . In this