Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways.We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC.NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting.The clinical prognostic value of NRP1 was determined by Kaplan–Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors.These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.The online version contains supplementary material available at 10.1186/s13058-022-01501-7. Breast cancer (BrCa) is a heterogeneous disease, with genomic profiling studies having identified five major human breast cancer intrinsic subtypes according to PAM50 classification , which differ in their molecular profiles, incidence and prognosis . The mosThe claudin-low subtype is most enriched for basal-like tumors (51.7%), and 25–39% of triple-negative tumors are claudin-low . While hNeuropilin-1 (NRP1) is a pleiotropic transmembrane co-receptor protein critical in embryonic development of neurological and vascular systems . It has We investigated the prognostic value, expression and function of NRP1 across BrCa subtypes. We report that high NRP1 expression predicts shortened time to disease relapse and metastasis in ER-negative patient cohorts and is associated specifically with the claudin-low subtype. NRP1 knockdown in claudin-low cell lines led to significant reduction in cell proliferation and growth of orthotopic claudin-low xenografts. NRP1 over-expression associated with the most de-differentiated claudin-low tumors and was required to maintain high expression of ZEB1 and the mammary stem cell marker ITGA6. Targeted inhibition with an NRP1 monoclonal antibody reduced spheroid-forming capacity and potently suppressed tumor growth in an orthotopic claudin-low TNBC xenograft model. Finally, we show that NRP1 acts as a central hub for the aberrant activation of the RAS-MAPK pathway via EGFR and PDGFR activation to drive aggressive tumor progression, a hallmark of all claudin-low subgroups. These data identify NRP1 as a key driver of the claudin-low phenotype and support further testing of NRP1 inhibitors for improved control of claudin-low tumor progression.NRP1 expression correlation with relapse-free survival (RFS) and distant metastasis-free survival (DMFS) was analyzed using Kaplan–Meier Plotter software  with the following non-default settings selected; Affymetrix ID / Gene symbol; 212298_at, Split patients by; upper quartile, Survival; RFS (4934) or DMFS (2767), Probe set options; JetSet best probe set, ER status—array (n = 7535); all, ER positive (5526) or ER negative (2009) . NR. NR26]. The Prediction Analysis of Microarrays (PAM) R package (pamr) was used to classify the three claudin-low subtypes using the ‘nearest shrunken centroids’ algorithm as reported by Pommier et al. . The preMDA-MB-231, BT-549, MCF-7, T47D, HS578T and SUM159 BrCa cell lines were sourced from the American Type Culture Collection . MDA-MB-468, BT-20, MDA-MB-361, BT474, SKBR3 and HCC1569 cells were obtained from the ATCC and transferred from Lombardi Cancer Center, USA, by Prof. Erik Thompson (QUT). All cell lines were authenticated by short tandem repeat analysis at the Genomics Research Centre . SKBR3, HCC1569 and T47D cells were cultured in RPMI 1630 (Gibco) supplemented with 10% FBS (Gibco). MDA-MB-231, SUM159P, HS578T, BT-549, MDA-MB-468, MDA-MB-361 and BT474 cells were cultured in DMEM (Gibco) with 10% FBS. MCF-7 cells were cultured in DMEM (Gibco) with 10% FBS and 10 ug/mL insulin (Gibco).Cells were harvested in lysis buffer  containing protease and phosphatase inhibitors  on ice for 30 min. Protein concentration was determined by the Pierce BCA Protein Assay Kit (Bio-Rad). Protein samples were prepared using Bolt LDS Sample Buffer and Reducing Agent (Thermo Fisher). Samples were heated at 70 °C for 10 min and separated on 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Thermo Fisher). Proteins were transferred onto a nitrocellulose membrane using the Mini Blot Module (Thermo Fisher Scientific). Membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% non-fat powdered skim milk for one hour, then incubated in primary antibody overnight as follows; NRP1 , GAPDH , EGFR , phospho-EGFR , γ-tubulin , ZEB1 , p42/44 , phospho-p42/44 , ITGA6 , PDGFR  or phospho-PDGFR  according to manufacturer’s instructions. After washing with TBS-T, horseradish peroxidase-conjugated secondary antibodies were applied for one hour at room temperature in 5% skim milk and immunodetection performed using Immobilon TM Western Chemiluminescent HRP Substrate (Millipore). Chemiluminescent signal was visualized using the Konica SRX-101A film processor or Chemidoc Gel Imaging System (BioRad). Densitometry was performed using Image Studio Lite software.GTGCT), ZEB1 , ITGA6 .RNA was extracted with the Direct-zol RNA miniprep kit (Zymo Research) before reverse transcription with the SensiFAST cDNA Synthesis kit (Bioline). Quantitative PCR was performed using SYBR Green (Thermo Fisher) using QuantStudio 6 Real-Time PCR System (Thermo Fisher). Gene expression was determined by the comparative Ct method, and normalized to the housekeeping gene RPL32. Expression in NRP1 knockdown samples was normalized to NT control. The following primer sequences (5’-3’) were used: NRP1 . Cells were neutralized with PBS/5% FBS and resuspended to 106 cells/ml. Anti-human APC-conjugated NRP1 antibody  or IgG-APC control  was added and cells incubated on ice for 45 min. Cells were centrifuged, resuspended in PBS/5% FBS and propidium iodide (100ug/ml) added. Fluorescence compensation and analysis was performed on a BD Accuri C6. Results were analyzed with Kaluza software. The X-axis parameter Median  was used to represent fluorescence intensity. For CD44/CD24 based cell sorting, 20 × 106 cells were grown to 80% confluence, washed twice in PBS and detached with Accutase (Thermo Fisher). After washing and resuspension (106 cells/ml) in PBS/5% FBS, the cell suspension was incubated with anti-human CD44-Alexa Fluor 488 ) and CD24-APC  for 1 h on ice, then washed three times in PBS/5% FBS. Propidium iodide  was added to the cells immediately before loading on a MoFlo Astrios Cell Sorter (Beckman Coulter) to allow for viable cell gating.For NRP1 expression analysis, cells at 80% confluence in 75 cmBrCa cells were reverse transfected using Lipofectamine RNAiMax reagent (Thermo Fisher Scientific) and 5 nM Silencer Select siRNA (Ambion) according to manufacturer’s instructions. The following NRP1 siRNA sequences were used : siNRP1 ; cagccuuCell viability was measured at 0, 1, 2, 4 and 7 days post-siRNA treatment. A total of 5,000 cells were plated per well of a 96-well plate after mixing with transfection solution as described. Viability was assessed using the CyQuant Direct Cell Proliferation Assay (Life Technologies) or Incucyte S3 (Essenbioscience) according to manufacturer’s instructions. For day 0 measurement, cell viability was quantified 6 h after cells were plated.https://cellprofiler.org/) based on spheroid diameter ≥ 50 μm or ≥ 18 pixels.Spheroid formation assays were carried as described previously , 30. BriPrkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from the Animal Resources Centre (ARC), Western Australia, and acclimatized for one week at the Biological Resources Facility (BRF), Translational Research Institute, before beginning experiments. Mice were maintained on standard irradiated rat and mouse diet  and provided a 12 h light/12 h dark cycle. Twice a week, mice received sunflower seeds as a supplement. Mouse health was monitored at least three times weekly by researchers and daily by BRF staff. All procedures were performed in a sterile laminar flow cabinet.All animal studies were carried out with approval from the University of Queensland Animal Ethics Committee . Eight-week-old female NSG (NOD.Cg-6 SUM159 cells constitutively expressing luciferase (SUM159luc) in 30 µL Matrigel (Corning) were injected into the left inguinal mammary fat pad of 8-week-old female NOD SCID gamma (NSG) mice. To test the effects of shRNA NRP1 knockdown, SUM159luc transfected with shNRP1 or shNT were used for injection (10 mice per group). Mice in the shRNA study were allowed to progress to ethical endpoint (tumor volume 1000 mm3). To test the effects of the NRP1 inhibitor Vesencumab, mice injected with SUM159luc cells were randomly allocated to IgG control or Vesencumab treatment groups , for 7 weeks. Bioluminescence imaging of tumors was performed with the IVIS Spectrum In Vivo Imaging System (Perkin Elmer) one-week post-xenografting to confirm presence of live tumor cells. Tumor size was measured by digital caliper twice weekly until endpoint, when bioluminescence imaging of tumors was repeated prior to tissue collection. Vesencumab and IgG control were provided by Genentech Inc.1 × 100C for 16 h as follows; NRP1 , Ki67 , CD31  and phospho-p42/44 , with Ki67, CD31 and phospho-p42/44 antibodies diluted according to manufacturer’s instructions. Slides were washed, incubated with EnVision + Dual Link System HRP (Agilent) for 30 min, washed again and developed in Liquid DAB + Substrate Chromogen System (Agilent). Slides were counterstained with Mayer’s hematoxylin, dehydrated and mounted in DPX. Slides were scanned by the Panoramic Digital Slide Scanner (3DHISTECH) and analyzed with CaseViewer software using QuantCenter (3DHISTECH). The PatternQuant module was used to discriminate epithelial, stromal and necrotic compartments. DensitoQuant  or NuclearQuant  modules were used for quantification of IHC staining. For NRP1 IHC quantification, the following DensitoQuant settings were used: Detection , Score . For phospho p42/44 quantification, the following NuclearQuant settings were used: Nucleus Detection , Nucleus Filters , Score .Paraffin sections were dewaxed, rehydrated and then underwent antigen retrieval in 1 mM EDTA buffer (pH 9) in a microwave oven (700 W) for 16 min. Endogenous peroxidase block was performed for 15 min in 3% hydrogen peroxide, followed by 3 washes of 10 min each with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T). Sections were blocked for 30 min with CAS-block reagent (Zymed Laboratory), followed by 3 PBS-T washes. Slides were incubated with the following primary antibodies diluted in Dako antibody diluent at 4 Cells were seeded into T25 flasks and serum-starved for 16 h when they reached 70% confluence. Cells were pre-incubated with Vesencumab or IgG control (50 μg/mL) for 1 h followed by receptor activation by addition of culture medium containing 20% FBS for 60 min. For NRP1 knockdown study, cells were transfected and seeded as described above. Two days post-transfection, cells were serum starved for 24 h followed by receptor activation by addition of culture medium containing 20% FBS for 0, 10 or 60 min. Protein lysates were then collected and antibody array binding performed according to manufacturer’s instructions . Antibody membranes were scanned by ChemiDoc (Bio-Rad) and densitometry performed using Image Lab software (Bio-Rad).Differences between two groups were compared using unpaired Student’s t test. For multiple group comparisons, one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used; for survival analysis, log-rank (Mantel-Cox) test was used to assess the significant differences among treatment and control groups. For in silico analyses, Chi-square test was used to assess the significance between subtypes, and linear regression analysis was used to calculate the significance between NRP1 expression and gene score. Tumor Control Index (TCI) was used to estimate the tumor size for one mouse  which was culled early for health reasons before to reaching ethical tumor volume endpoint. The Srivastava Lab kindly provided the VBA macro with graphical user interface (SL TCI) that calculates TCI scores, based on tumor rejection, regression and stability scores .p = 0.0028) was observed between high NRP1 expression (Q4) and shorter overall survival time  was used to interrogate a combined dataset comprising 50 independent BrCa patient cohorts [p = 0.43; HR 1.05) or distant metastasis-free survival (DMFS)  across an unselected cohort of patient tumors , while no significant difference was seen in ER-positive patients . For DMFS, high NRP1 levels were most strongly associated with poor outcome in ER-negative patients , whereby ER-negative patients with NRP1 expression in the upper quartile had a relatively short time to distant metastasis . Hence, elevated levels of NRP1 expression were indicative of poor BrCa patient outcomes, particularly in ER-negative patient cohorts.When patients were stratified according to positive or negative ER status, NRP1 association with shorter time to RFS was more pronounced in ER-negative patients  tumors compared to non-core claudin-low and non-claudin-low tumors  identifies the core cell–cell adhesion, EMT and stem cell-like pathways that are hallmarks of the claudin-low subtype , 10. NRPors Fig. a, e. The2 cohort , suggestNRP1 expression  was reached. NRP1 expression was suppressed in shNRP1 cells at the time of injection, as confirmed by Western blotting in the cells remaining after xenografting  has recently been defined . In theills Fig. g [11, 25lls Fig. H. Assess+/CD24low subpopulation can reportedly enrich for a subpopulation of cells with self-renewal potential [+/CD24low population, SUM159 cells were sorted into CD44+/CD24low and CD44+/CD24high populations. Concomitant with a reduction in ZEB1, NRP1 levels were slightly reduced in CD44+/CD24high cells , qPCR analysis of NRP1 in CD44+/CD24− and CD44+/CD24+ MDA-MB-231 subpopulations recapitulated the results obtained using SUM159 cells , which targets the extracellular VEGF-binding b1/b2 domain of NRP1, versus IgG control (50 µg/ml each) . Vesencu6 luciferase-expressing SUM159Luc cells into the inguinal mammary fat pad, female NSG mice were treated twice weekly with Vesencumab or IgG . At study endpoint 7 weeks after tumor inoculation, tumor-derived luciferase signal was greater in the IgG control versus Vesencumab group  of triple-negative tumors classify as claudin-low. We report high NRP1 expression to be associated specifically with the claudin-low molecular subtype of breast cancer. This correlation was not due to immune or stromal cell infiltration in claudin-low tumors, as NRP1 expression correlated with a ‘core’ claudin-low signature enriched in EMT and cancer stem cell markers that characterize the claudin-low subtype .+/CD24low population and conferred spheroid-initiating potential to claudin-low cells. Consistent with a known role in mediating integrin function in breast and other cell types, NRP1 was required to maintain expression of the mammary stem cell marker ITGA6 (CD49f) [NRP1 expression associated more strongly with the claudin-low up, rather than down, gene signature score, which is enriched in mesenchymal and stem cell markers. NRP1 expression was highest in CL1 subtype tumors, which most closely resemble mammary stem cells . In the  (CD49f) –45. Thes (CD49f) .The NRP1-targeted antibody Vesencumab was able to potently inhibit the growth of SUM159 orthotopic xenografts, whereby the mean volume of Vesencumab-treated tumors at the 7-week study endpoint was 12.8-fold smaller than IgG treated tumors. The main effects of Vesencumab were likely due to direct anti-tumor effects on tumor cells and largely independent of changes to angiogenesis, as no difference in CD31-positive intratumoral vasculature was observed between Vesencumab and IgG treated tumors. In our model, Vesencumab was administered at the time of tumor xenografting until study endpoint to establish the effects of NRP1 inhibition on tumor latency and growth kinetics, as suggested by its association with a mammary stem cell population. In future experiments, it will be important to determine the tumor-suppressive potential of Vesencumab when administered to animals with established tumors or after tumor resection, to more closely resemble the timing of therapeutic intervention in future clinical trial testing.As all the claudin-low cell lines used in this study were basal-like, further studies assessing the efficacy of NRP1 inhibition across claudin-low models of different intrinsic subtypes will also be of interest. As high NRP1 expression was particularly associated with poor prognosis in ER negative tumors, we suggest that the greatest clinical utility of NRP1 inhibition is likely to be in patients with claudin-low TNBC.Evidence is accumulating that aberrant activation of the RAS/MAPK pathway is a key mechanism capable of driving transition to a claudin-low phenotype , 47. DesNRP1 has recently been implicated in resistance to cancer therapies including oncogene-targeted therapies and chemotherapy through activation of bypass survival pathways, including receptor-tyrosine kinase pathways such as HER2, EGFR and IGF1R , 21, 43.There is a shortage of durable targeted therapies available to treat TNBC, which lack expression of the clinically actionable estrogen/progesterone receptors and HER2 amplification. Our findings identify NRP1 over-expression as a feature of claudin-low breast cancers, a substantial proportion of TNBC. Taken together, these data strongly implicate NRP1 in claudin-low tumor progression and provide the preclinical rationale for future studies assessing NRP1 inhibition as a novel therapeutic strategy for this aggressive BrCa subtype.Additional file 1. Supplementary figures 1–6.


Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly or indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal  and systemic antibacterial immunity of invertebrates.Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp ( Marsupenaeus japonicus) and found that it played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity and directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. This study revealed the different functions of FOXO in the innate antibacterial immunity of invertebrates.Shrimp aquaculture is one of the world’s fastest growing industries for producing animal proteins and has made a significant contribution to meeting the worldwide increased demand for animal proteins. However, disease outbreaks in aquaculture result in large economic losses to the industry. Studies of shrimp immune mechanisms could provide new strategies for disease prevention and control. The forkhead box transcription factor O family proteins (FOXOs) are involved in various critical biological process of organisms. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from kuruma shrimp ( Caenorhabditis elegans  × 100%. The phagocytotic index was defined as [the number of total bacteria/ the number of all cells observed or tested] × 100%. To confirm the above results, the phagocytosis rate of hemocytes were also assayed using flow cytometry . The FITC-labeled bacteria (1 × 107) were injected intramuscularly into shrimp. Then, the hemocytes of 5 shrimp were collected at 2 h post injection and washing twice with sterile PBS by centrifugation. No staining was performed for the whole hemocytes in the phagocytotic analysis, other than the labeling bacteria with FITC. Before flow cytometry analysis, the whole system of flow cytometry was calibrated and the gates (cell size) was set in flow cytometry to remove impurities and hemocyte debris. Afterwards the hemocytes were collected by flow cytometry  and used the fluorescence detection channel (488 nm) to detect FITC fluorescence signal. About 3000 hemocytes were collected in each group. The fluorescence intensity of the collected cells was screened and the phagocytosed hemocytes containing bacteria (R2) were separated from non-phagocytosed hemocytes (R3) by fluorescence signal. Each group of flow cytometry analysis contained 3000 hemocytes and 5 repetitions were performed. The phagocytosis rate was analyzed by IDEAS Application v6.0 software.The bacteria in logarithmic growth phase were collected by centrifugation at 5000 × g for 5 min at 4°C and washed twice with PBS. Fluorescent isothiocyanate (FITC)  was used to label d method . The FITThe hemolymph was extracted from the ventral sinus of shrimp, and hemocytes were collected using the above-mentioned method. The collected hemocytes were washed three times with PBS and were dropped on polylysine coated slides and incubated for 3 h. After treatment with 0.2% Triton X-100, the hemocytes were washed with PBS three times, and then blocked with 3% BSA (PBS dilution) at 37°C for 1 h. The hemocytes on the slide were incubated with 3% BSA-diluted antibody (1:50 dilution) and incubated overnight at 4°C. The next day, the hemocytes were incubated with 3% BSA-diluted goat anti-rabbit Alexa 488 or antirabbit IgG Alexa-546 (diluted at 1:500) for 1 h, after washed five times with PBS. Then, the hemocytes were washed five times with PBS and stained using DAPI for 10 minutes. Finally, after three washes with PBS, the slide was observed under a fluorescence microscope . WCIF ImageJ software  was used to analyze the colocalization of FOXO and DAPI-stained nuclei in the hemocytes.V. anguillarum (3 × 107 CFU) was injected into shrimp, and hemocytes were collected using the above-mentioned method for a fluorescent immunocytochemistry experiment. The co-localization of bacteria with RAB5 or RAB7 was observed under a fluorescence microscope  and analyzed using WCIF ImageJ software [After FITC staining, software .t-test for paired comparisons or one-way ANOVA for multiple comparisons. Asterisks in figures indicate statistical significance . The different lowercase letters indicate significant differences (p < 0.05) in the ANOVA analysis. The nonparametric test, Mann-Whitney U test was also used for the un-normally distributed data. The survival rate was calculated and the survival curves are presented as Kaplan–Meier plots and the statistically using a log-rank test. All statistical analysis were produced using GraphPad 8.0 data view software. Western blotting bands were analyzed based on three independent replicates using ImageJ software .Data were presented as the mean ± standard deviation (SD) of at least three replicates. Significance differences were analyzed using Student’s S1 Fig(A) Comparison of the FOXO domain architectures and AKT/PKB phosphorylation sites between M. japonicus and Homo sapiens. The FOXO amino acid sequence of Homo sapiens were obtained from GenBank . (B) Phylogenetic tree of FOXOs from different species. The FOXO sequences of different species were obtained from GenBank, and the NJ tree was established using MEGA 6.0. The results were repeated 1000 times by bootstrapping. FOXO of M. japonicus is marked by a red triangle.(TIF)Click here for additional data file.S2 FigThe posttranslational modification sites of FOXOs are shown in the alignment following previous reports on FOXOs. NLS: nuclear localization signal; NES: nuclear export sequence. CBP: cyclic-AMP responsive element binding (CREB)-binding protein; JNK: c-JUN N-terminal kinase; ERK: extracellular regulated protein kinase; SIRT1: Sirtuin1.(TIF)Click here for additional data file.S3 Fig(A) Efficiency of Foxo-RNAi in hemocytes and intestines analyzed using qPCR. (B) mRNA expression of Foxk2 in Foxo-RNAi shrimp detected using qPCR. (C) mRNA expression of Foxn3 in Foxo-RNAi shrimp analyzed using qPCR. (D) Western blotting analysis of FOXO protein in the cytoplasm and nuclei of intestine cells in Foxo-knockdown shrimp. (D’) The results of statistical analysis of three replicates for panel D. (E) The nuclear translocation of FOXO protein in the hemocytes from Foxo-knockdown shrimp at 2 h post V. anguillarum challenge analyzed by fluorescent immunocytochemical assay. Scale bar = 20 μm. (E’) Statistical analysis of FOXO nuclear translocation. WCIF ImageJ software was used to analyze co-localization by detecting the fluorescence intensity ratio of anti-FOXO (green) and DAPI-stained nuclei (blue) in hemocytes.(TIF)Click here for additional data file.S4 FigAlf-E1 genomic sequence were marked. with green. Transcriptional start site marked with red.The FOXO binding sites of (TIF)Click here for additional data file.S5 Fig The mRNA expression patterns of Rab5 (A) and Rab7 (B), as analyzed by qPCR.  Expression patterns of RAB5 and RAB7 proteins was analyzed using western blotting. The results of statistical analysis of three replicates are shown in the lower panels of (C) and (D). The relative expression levels of RAB5 or RAB7 normalized to that of β-actin were expressed as the mean ± SD, and the value of the control shrimp was set as 1. (E) Efficiency of Rab5 and Rab7 RNAi, as determined using western blotting and qPCR. (F) The survival rate of shrimp after knockdown of Rab5 or Rab7 following V. anguillarum infection. The survival rate of each group was calculated and the survival curves are presented as Kaplan–Meier plots.(TIF)Click here for additional data file.S6 FigA) The mRNA expression level of the Rab5 and Rab7 in intestine of Foxo-RNAi shrimp with and without bacterial infection determined by qPCR. (B) The protein expression level of the RAB5 and RAB7 in intestine of the shrimp determined by western blotting. (B’), The results of statistical analysis of three replicates of panel (B).((TIF)Click here for additional data file.


Background: Over 50 million cases of COVID-19 have been confirmed globally as of November 2020. Evidence is rapidly emerging on the epidemiology of COVID-19, and its impact on individuals and potential burden on health services and society. Between 10–35% of people with COVID-19 may experience post-acute long Covid. This currently equates to between 8,129 and 28,453 people in Scotland. Some of these people will require rehabilitation to support their recovery. Currently, we do not know how to optimally configure community rehabilitation services for people with long Covid.Methods: This national survey aimed to provide a detailed description of current community rehabilitation provision for people with long Covid in Scotland. We developed, piloted, and conducted a national electronic survey of current community rehabilitation service provision for people presenting with long Covid symptomatology. Our sample were the Allied Health Professions Directors of all 14 territorial NHS Health Boards in Scotland. Fixed response and narrative data were analysed descriptively.Results: Responses were received from all respondents (14/14), enabling a national picture to be gained. Almost all Health Boards (13/14) currently deliver rehabilitation for people with long Covid within pre-existing services.Fatigue (11/14) and respiratory conditions (9/14) were the two most common presenting problems of patients. Most long Covid community rehabilitation services are delivered through a combination of face-to-face and digital contact (13/14).Conclusions: Community rehabilitation for people with long Covid is an emerging reality. This survey provides a national picture of current community rehabilitation for people with long Covid. We do not know how community rehabilitation can be optimally delivered for this population. This is vital as community rehabilitation services were already under pressure prior to the emergence of COVID-19. Further research is urgently required to investigate the implementation, outcomes and cost-effectiveness of differing models of community rehabilitation for this patient population. Among the consequences of COVID-19 is the impact of long Covid, where individuals are left with debilitating symptoms after the initial acute phase of infection2. To date there is no agreed definition of what constitutes long Covid, however it has been proposed that it is when individuals have symptoms extending three weeks beyond onset (post-acute COVID-19) and 12 weeks beyond onset (chronic COVID-19)3. Long Covid does not only affect people who were severely ill, but also people with milder symptoms, and those who were not hospitalised4.Since the initial emergency of COVID-19 in Wuhan, Hubei Province, China in December 2019, the disease has rapidly spread across the globe, with more than 50 million cases now confirmed globally (9 November 2020)5. Globally this equates to between five and 17.5 million people who may experience debilitating aftereffects of the infection. Within Scotland (as of 15 November 2020) 1,086,353 people have been tested for COVID-19 with81,294 people testing positive. Using this data, we can estimate that between 8,129 and 28,453 people may have post-acute COVID-19, with around 812 people remaining significantly unwell at 12-weeks, commonly due to organ damage6. Consequently, long Covid has received considerable media attention in Scotland and beyond, with action groups calling for more support for symptom management7. As around 10% of people who experience mild COVID-19 may go on to experience long-term symptoms8, it is important that rehabilitation is accessible to those presenting in community settings as well as being discharged from hospital.It is estimated that 10–35% of people infected with COVID-19 may experience post-acute long Covid9. The presentation and severity of these symptoms are variable. Several people who have long Covid report a non-linear journey of recovery and describe their symptoms as moving around their body, such that as one symptom abates, another appears9.Reported symptoms of long Covid vary widely. They commonly include respiratory, cardiopulmonary, neurological, musculoskeletal and mental wellbeing sequelae, as well as fatigue and loss of taste and smell10. Rehabilitation for long Covid is in its infancy. We do not currently know how rehabilitation can be optimally delivered for people with long Covid. Findings from a recent living systematic review found most publications have been expert opinion about how rehabilitation for long Covid should be delivered, indicating that high-quality research is required11. Understanding how to optimally deliver long Covid community rehabilitation is vital, as rehabilitation services need to cope with additional demand while continuing to provide rehabilitation for other, often vulnerable, patient populations12.A currently unknown number of people with long Covid will require rehabilitation to support their recovery and increase their quality of life. As with other long-term conditions, rehabilitation for people with long Covid should be multidisciplinary, comprehensive, and tailored to individuals’ needs, in order to maximise function, quality of life and participation in societyThis paper reports a recently conducted national survey of current community rehabilitation provision for people with long Covid. The aim of the survey was to provide a detailed description of current community rehabilitation provision for people with long Covid across Scotland. We believe it to be the first published national survey describing long Covid rehabilitation models of practice. The survey is the first step in a programme of research to investigate how community rehabilitation can be optimally delivered for people experiencing long Covid.Jisc online survey tool we developed and conducted a national electronic survey for the Directors of Allied Health Professions of all 14 territorial NHS Health Boards in Scotland. The aim of the survey was to discover their current service provision for rehabilitation of people presenting with long Covid symptomatology in the community. The survey is reported in keeping with recommended reporting guidance for surveys13.Using the14:An initial draft survey was developed by the study authors. This incorporated fixed item and narrative response survey questions, informed by the TIDieR Intervention Description checklistHow? How is long Covid rehabilitation delivered in your board area?Why? What are the main problems that patients require rehabilitation for?What is provided? Please describe the service as fully as you can.Who provides? What professional groups are involved in delivering long Covid rehabilitation in your board area?How/where is it provided? How do patients access long Covid rehabilitation in your board area?When and How much? Can you describe the timing and duration of typical long Covid rehabilitation in your board area?final questionnaire used in this study is available as extended data.We conducted a small pilot of the initial survey content with the Scottish Government’s Professional Advisor for Rehabilitation, the National Clinical Lead for Digital Health and Care, the Allied Health Professions’ Improvement Advisor for Healthcare Improvement Scotland, and the director of services of Chest Heart and Stroke Scotland. Minor changes to the survey wording were made based on their feedback. A copy of theHealthcare in Scotland is primarily delivered through NHS Scotland’s 14 territorial Health Boards. Each Health Board covers a separate region. Together they cover the entire Scottish population. They are responsible for the protection and improvement of the health of the people in their region and the delivery of healthcare services. Each Health Board has a Director of Allied Health Professions. We invited all 14 Directors of Allied Health Professions to participate in this survey via an emailed letter. To minimise the potential of attrition bias, the letter from the study authors containing a link to the survey was emailed by the Scottish Government’s Professional Adviser for Rehabilitation to each of the Directors.The online survey was launched on 14 October 2020 and closed on 6 November 2020.Fixed response item data  were analysed descriptively. Narrative responses to open ended questions  were mostly short statements and insufficient to conduct for a formal thematic analysis or other qualitative method. Instead two members of the research team  reviewed responses and descriptively report them in the paper where they related to the study results.As the study surveyed current practice it did not require research ethics approval by the NHS. Data was stored on password protected University servers in compliance with European Union General Data Protection Regulation (GDPR) standards of data protection and storage. The covering letter to potential participants explained the reasons for the survey and that their anonymised responses may be published. Informed consent to participate and for the publication of results was implied through their return of the study questionnaire.underlying data.We received responses from all 14 Directors of Allied Health Professions, enabling a national picture of community rehabilitation service delivery for people with long Covid to be gained. An anonymised copy of all survey response data is available asAlmost all Health Boards (13/14) are currently delivering rehabilitation for people with long Covid within pre-existing services. One Health Board has developed a new service for people requiring long Covid rehabilitation, and another is currently developing a new service. Data on the numbers of patients who have received long Covid rehabilitation to date were not available from most respondents (12/14), indicating that routine rehabilitation data collection methods are not yet universally established. In services that were able to provide referral number data (2/14), one respondent  stated that they had received a referral for one patient in total, while the Health Board with a specialist long Covid service stated that they had received 95 referrals in eight weeks.Respondents reported that the main symptoms requiring rehabilitation interventions were fatigue (11/14), respiratory conditions (9/14), musculoskeletal conditions (6/14), mental health (5/14), and neurological impairments (4/14). One respondent stated that patients who were referred to their service experienced fatigue (86%), respiratory symptoms (67%), reduced mobility/exercise tolerance (60%), low mood, anxiety, depression (43%), cardiac symptoms (24%), sleep disturbance (24%), and weight management concerns (12%).Respondents did not describe the therapeutic content of long Covid rehabilitation in any detail, referring instead to the professions that were involved in delivery of the service (see below). One respondent described their service providing energy conservation advice and assessment of aids and adaptations. Another respondent described their service as providing fatigue management, confidence building, muscle strengthening, anxiety management, nutritional advice, breathing re-education, and activities to support individuals to regain function. Another said their service used a combination of pulmonary rehabilitation and community reablement. A final respondent described their service as providing individualised goal setting based on symptomatic presentation.Community rehabilitation service provision for people with long Covid is multidisciplinary. Almost all services (13/14) include occupational therapy and physiotherapy. Many include dietetics (11/14) and speech and language therapy (9/14). Half include psychology input (7/14). In addition, three services reported being able to refer to, or having the involvement of differing resources including post intensive treatment nursing teams, therapy assistant practitioners, outpatient services for people with neurological conditions, spiritual care teams, and specialist rehabilitation medical consultant services.Most long Covid rehabilitation services are delivered through a combination of face-to-face and digital contact (13/14). While precise numbers were not available, respondents reported large variations in the percentage of rehabilitation being delivered through the different forms of delivery, depending on clinical need. One respondent reported that their primary delivery route was digital. Another reported only delivering long Covid rehabilitation face-to-face, with no digital service.Almost all respondents (13/14) reported patients being able to access long Covid rehabilitation through either hospital or GP referral. Many respondents stated that patients could also access long Covid rehabilitation through self-referral (11/14). Some respondents (3/14) stated other routes of access to long Covid rehabilitation including interdisciplinary referrals from other allied health professionals and social care, as well as referrals from informal carers. Responses on typical duration of rehabilitation were limited. Three respondents stated it was dependent on the individuals’ needs.15, our findings demonstrate that community rehabilitation for people with long Covid is an emerging reality and is being provided across Scotland. Community rehabilitation for people with long Covid is currently being delivered predominantly by multidisciplinary teams of allied health professionals, with other specialists available as required. This is in keeping with community rehabilitation for other long-term conditions16. We have found variation in the modes in which long Covid rehabilitation is currently being delivered  and provided (integrated services/new services) in Scotland. Symptoms that people with long Covid are presenting with to rehabilitation services are in keeping with the literature to date and provide an indication of the skill-mix and expertise required within a long Covid rehabilitation service. Irrespective of the number of patients requiring rehabilitation for long Covid within a particular health board area, services will need to be able to provide appropriate and accessible rehabilitation, responsive to the diverse symptomatology and wider impact of the condition. Mode of delivery will be compounded by ongoing physical distancing measures.Despite some expert opinion that referral to community rehabilitation is not required for many people who have had COVID-1918, and the one Health Board in our survey that reported developing a new long Covid specific service, described providing a predominantly digital service. Data on rehabilitation services in Scotland is not routinely collected, so detailed information on the numbers of referrals of people experiencing long Covid, the problems with which they were presenting, duration of rehabilitation, and specific interventions delivered was unavailable.This study has several limitations. We do not yet know which modes of delivery are most appropriate for this patient population. Innovative tele-health services are beginning to be proposed for this patient groupThis survey provides a national picture of current community rehabilitation for people with long Covid symptomatology. To the best of our knowledge this is the first national survey of its kind. There is still lots to learn about current practice. While data on numbers of referrals, and content and duration of rehabilitation was requested, this information was not available to most of the respondents. A description of current services also does not provide any information on the effectiveness of the community rehabilitation service for people with diverse presentations, or of its perceived acceptability by its recipients. Detailed data on services and their recipients is vital and urgently required, to guide effective and efficient clinical practice and service planning and delivery. Following up this survey with qualitative interviews or a focus group would have provided richer and more in-depth information, however these options were not feasible within the timeframe that was available. Further research into long covid rehabilitation is now being conducted by the research team which includes interview and focus groups methods and we are confident that these methods will enhance our understanding of current community rehabilitation for people experience long covid.21. How to optimise delivery of community rehabilitation is unknown, but vital to determine, given that rehabilitation services need to cope with additional COVID-19 demand whilst continuing to provide rehabilitation for other, often vulnerable, patient populations21.Several UK bodies and individuals have published expert opinion recommending a stepped, needs-based community rehabilitation approach incorporating information provision, self-management support and specialist services as required. They also recommend that rehabilitation should be individualised, progressive and utilise digital solutions22, which is provided in different ways according to clinical need, geographical location and financial costs. This complexity is further exacerbated when treating people with long Covid where the impact of the clinical sequalae is still unknown. While community rehabilitation has been routinely provided within the NHS in Scotland for many years, there are many unknowns regarding the delivery of community rehabilitation for people with long Covid. Therefore, research is urgently required to evaluate which models of community rehabilitation work, in what circumstances, and with whom.Community rehabilitation is a complex interventionThis paper reports the findings of a national survey of current community rehabilitation provision for people with long Covid in Scotland. Almost all current services are providing a community rehabilitation response within current service provision. There is variation in the way in which these services are provided. Some information was unavailable due to the lack of routine data collection. With growing numbers of people presenting with symptoms of long Covid, further research is urgently required to investigate the implementation, outcomes and cost-effectiveness of differing models of community rehabilitation for this patient population.http://hdl.handle.net/11667/164DataSTORRE: Stirling Online Repository for Research Data. Survey Data for Long covid rehabilitation study.This project contains the following underlying data-   Anonymised survey responses in .xlsx formatCreative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of thehttp://hdl.handle.net/11667/165DataSTORRE: Stirling Online Repository for Research Data. Survey Data for Long covid rehabilitation study.This project contains the following underlying data-   A copy of the survey sent to participants in .pdf formatCreative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the I am happy that the authors have addressed my comments, I have nothing further to add.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:RehabilitationI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Nothing to add.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Community based occupational therapy with older people and people with chronic conditions.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I found this article an interesting and important read. I was wondering how a person with long COVID is identified - especially if their symptoms persist over 3 months after their COVID infection, and whether or not their access to rehabilitation would be dependent on them complaining of symptoms to someone who could refer such as their GP. This might mean that there are not the full numbers of people with long COVID who would be presenting to rehabilitation services which would limit the feedback that such services could provide in this survey. I was also interested that it was only the lead allied health people who were surveyed (n=14) since these people might not have a full idea of what services are actually provided at the coal face, being in more management positions. It would be interesting to send a similar survey to all community rehabilitation health professionals to get more detailed information. I'm sure the rural/urban differences would be interesting to look at with a larger sample.  Having said that, all these limitations were adequately addressed by the authors. I'm sure there will be further work done in this area.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Community based occupational therapy with older people and people with chronic conditions.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr MacKenzie, Many thanks for your review and positive feedback. In response to another reviewer we have added a comment in the limitations of our revised paper, which highlights that some of our future research will investigate the issues that you raise. This is an important and timely piece of work which provides an overview, at a national level, of community based rehabilitation for people with long Covid, and key gaps in knowledge. Strengths of the work are the survey design i.e. using Surge guidelines for reporting and TIDIER as a structure for the questions with a combination of closed and open narrative questions; the team piloted the tool prior to use and involved key stakeholders in its development. It is a well written manuscript with use of key recent supporting references. There was an excellent overall response rate to the survey, and within the survey to the individual closed questions. The response to some open questions was good, and is absent for others. Overall this work is an important first step to inform service provision for managing people with long Covid.Items to consider addressing:Was there a plan to collate the responses to the narrative questions? and if so please add this to your data analysis section.Please clarify in your methods:Were there reminders to respondents to complete the questionnaire or a single invitation only?Did the team consider other ways of enhancing the responses to the narrative questions? Might their open approach be a limitation to their survey? Do they think further closed questions with options may have helped, or a qualitative interview/focus group follow up?In the discussion, first paragraph, you state that there was a wide variation in the numbers accessing services, this needs to be qualified as I understand from your results that this data was only available for 2/14 of the respondents so is largely unknown.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:RehabilitationI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dear Professor McDonough, Thank you for taking the time to review this paper and for your helpful comments. Our responses to your review are below. Items to consider addressing:Was there a plan to collate the responses to the narrative questions? and if so please add this to your data analysis section. RESPONSE: Narrative responses were mostly short statements and insufficient to conduct for a formal thematic analysis or other qualitative method. Instead two members of the research team  reviewed responses and descriptively report them in the paper where they related to the study results. We have added this clarification to the methods section of our paper.Please clarify in your methods:Were there reminders to respondents to complete the questionnaire or a single invitation only? RESPONSE: As stated in the methods section, under "data analysis" - reminders were sent to non respondents after 1 week.Did the team consider other ways of enhancing the responses to the narrative questions? Might their open approach be a limitation to their survey? Do they think further closed questions with options may have helped, or a qualitative interview/focus group follow up? RESPONSE: Following up this survey with qualitative interviews or a focus group would have provided richer and more in-depth information, however these options were not feasible within the timeframe that was available. Further research into long Covid rehabilitation is now being conducted by the research team which includes interview and focus groups methods and we are confident that these methods will enhance our understanding of current community rehabilitation for people experience long covid. We have added this clarification to the methods section of our paper.In the discussion, first paragraph, you state that there was a wide variation in the numbers accessing services, this needs to be qualified as I understand from your results that this data was only available for 2/14 of the respondents so is largely unknown. RESPONSE: We have removed this sentence in response to your feedback.


Augmenting reality via head-mounted displays (HMD-AR) is an emerging technology in education. The interactivity provided by HMD-AR devices is particularly promising for learning, but presents a challenge to human activity recognition, especially with children. Recent technological advances regarding speech and gesture recognition concerning Microsoft’s HoloLens 2 may address this prevailing issue. In a within-subjects study with 47 elementary school children (2nd to 6th grade), we examined the usability of the HoloLens 2 using a standardized tutorial on multimodal interaction in AR. The overall system usability was rated “good”. However, several behavioral metrics indicated that specific interaction modes differed in their efficiency. The results are of major importance for the development of learning applications in HMD-AR as they partially deviate from previous findings. In particular, the well-functioning recognition of children’s voice commands that we observed represents a novelty. Furthermore, we found different interaction preferences in HMD-AR among the children. We also found the use of HMD-AR to have a positive effect on children’s activity-related achievement emotions. Overall, our findings can serve as a basis for determining general requirements, possibilities, and limitations of the implementation of educational HMD-AR environments in elementary school classrooms. Augmented reality (AR) is an emerging technology in education that enables real-time integration of real and virtual objects in the field of view ,2. The rFollowing Santos et al. , the oveMost educational AR-applications are designed for handheld display-devices like smartphones or tablets, while head-mounted display AR-devices (HMD-AR-devices) are used rarely . NeverthAccording to Yuen et al. , educatiHowever, the use of AR in education can be obstructed by technical issues and can require additional instruction . Hence, In order for AR-devices to exert their positive impact on information processing during learning, the handling and interaction with the device itself or with the virtual learning information offered must not itself lead to load on the learners as described in the previous Section. According to several reviews on AR in education, an often-reported issue concerning the practical use of AR-devices and applications in educational situations is the underwhelming usability ,36,37. TFor many applications, multimodal interfaces have long been recognized to be more robust, accurate, and preferred by users than unimodal ones. A major benefit is that users can freely choose their preferred modality combination . HoweverAnnounced innovations and improvements concerning the spatial positioning, speech, and gesture recognition for the successor model HoloLens 2 by Microsoft see  do not oThe HoloLens 2 offers various means to interact in AR. To describe these modes of interactions, we will focus on the action ‘selection of an AR-object’ from the tutorial that is pre-installed on the device see . The AR-Children are vulnerable and it is the responsibility of adults to protect them from possible harm. Technologies in research on children should therefore be applied very prudently. Particularly when using immersive technologies, such as AR and virtual reality (VR), special precautions should be taken . To ensuEvaluation of the overall usability of the HoloLens 2 as an HMD-AR-device;Comparison of the provided AR-interaction modes concerning their efficiency;Assessment of the children’s interaction preference in HMD-AR;Examination of the change in activity-related achievement emotions.Our aim is to assess the usability of Microsoft’s HoloLens 2 as the latest HMD-AR-device for the use with elementary school children. The device is not yet technically designed for use with children younger than 13 years: young children’s lower interpupillary distance might hamper the perception of virtual objects . TherefoWe invited 47 students  to participate in a laboratory study at the Saarland University. They took part in another study at the same day (either before or after attending this study), but the other study did not include the use of an HMD-AR-device. None of the children had previous experience with AR. In the beginning, we conducted test runs with four children , so n = 43 valid data sets were collected.The study was conducted using a within-subjects design. The independent variable was interaction mode, and the modes were modeled as different measuring points. The different multimodal AR-interaction modes provided by HoloLens 2  were compared regarding the dependent variables ‘mean number of attempts’ and ‘mean time’. For the children’s personal interaction preference in AR, we formed the variables ‘most favorite interaction mode’ and ‘least favorite interaction mode’. The most and the least favorite interaction mode can be ‘tap’, ‘air-tap’ or ‘voice command’. To investigate general effects of HMD-AR-usage on activity-related achievement emotions, we formed a pre- and a post-test variable for ‘enjoyment’, ‘boredom’ and ‘frustration’. To assess the overall device usability, we formed the variable ‘system usability score’.Due to the COVID-19 situation, only individual appointments with private journeys could be made. Prior to the start of the study, parents were informed about the investigation and gave their written consent for their children’s study participation. The procedure of the study was centered around a standardized tutorial on interaction in HMD-AR on the HoloLens 2 in German language. Before starting the tutorial, we assessed the children’s enjoyment, boredom and frustration. These activity-related achievement emotions are assumed to allow for inferences about motivation and interest ,33 vari. MoreoveFirst, we calculated the SU score. Subsequently, we compared the interaction modes with respect to their efficiency and evaluated the children’s interaction preferences in AR. Lastly, we examined the changes in the activity-related achievement emotions.The overall system usability was assessed using the variable ‘system usability score’. It is calculated as a descriptive measure from the children’s answers to the SUS-questionnaire. We used a five-point instead of the original ten-point-Likert scale to ease the rating process for the children and adjusted the score calculation accordingly. The scale is acceptably reliable (Cronbach’s Alpha = 0.76) for the sample. In addition, technical issues taken from the recorded tutorial videos that may impact the usability when wearing or using the device are described.The dependent variables ‘mean number of attempts’ and ‘mean time’ are compared for the three measurement points ‘tap’, ‘air-tap’ and ‘voice command’ using two respective non-parametric Friedman-tests for differences among repeated measures.The quantitative distributions of the children’s most and least favorite interaction mode in AR are reported. Reasons given for their decisions are presented.Changes regarding the activity-related achievement emotions were assessed by three Wilcoxon-signed-rank-tests for paired samples comparing the pre and posttest variables ‘enjoyment’, ‘boredom’ and ‘frustration’. Each emotion item of the pretest is based on a five-point Likert-scale . In the second questionnaire the children rated their current emotions in comparison to the previous measuring point (options: ‘much less’—‘a little less’—‘unchanged’—‘a little more’—‘much more’). Therefore, data transformation was applied. For further details see The ‘system usability score’ was calculated based on the children’s answers on the five-point Likert-scale with high ratings on regular items increasing the score and high ratings on inverse items decreasing the score following Brooke  see Doc. The medApart from minor  technical issues, one (that may impact the device usability) occurred frequently: In 19 out of 43 subjects (56%), the AR-objects moved away from the child’s hand as it approached the object. This occurred when the child had to move the body towards an object to reach it, but not when the child stood still when interacting with an object. For a demonstration of the issue see Chi-Square (2) = 72.29, p < 0.001) and the required time (Chi-Square (2) = 82.19, p < 0.001). Pairwise post-hoc comparisons (see The two non-parametric Friedman-tests of differences among repeated measures (for descriptive statistics see sons see  indicateAs shown in Z = p < 0.001), and a decrease in boredom (Z = p < 0.001) and frustration (Z = p < 0.001).The Wilcoxon-signed-rank-tests for paired samples see  revealedTo sum up, HoloLens 2 has proven to be an effective and appropriate HMD-AR-device to use with elementary school children, also confirmed by their subjective usability ratings. While children managed to use all provided interaction modes, the newly implemented direct ‘tap’-interaction turned out to be the best performing mode of interaction with virtual elements. Their reported most favorite interaction mode, however, was the ‘voice-command’-interaction. Although children were unfamiliar with HMD-AR-devices, the positive effects on activity-related achievement emotions suggest that educational settings may even benefit from using the HoloLens 2.The usability of the device was rated with an average system usability score of 80, resulting in the rating ‘good’ (scores greater than 82 indicate “excellent” usability) . Still, The frequently observed technical issue of AR objects moving away from children could stem from the device settings concerning the relative positioning of AR objects to the viewer, which seem to be designed for adults and their body dimensions. This did not occur in any of the adults that used the device. However, this is a preliminary statement without statistical validation as we did not investigate the occurrence of this issue comparing children with adults. It is assumed that the spatial position of the AR-objects in the tutorial is preset for adults and their physical appearance and that the children need to make a step towards an object to reach it. Yet, this causes the AR-objects to move away from the child as the device registers the spatial movement of the child and tries to maintain the relative position between spectator and AR-object. For a successful use of the device in educational situations with young children, the relative positioning of AR-objects should therefore either be scaled down to the length of children’s arms or absolute object positioning should be selected instead.The absence of physical inconveniences related to simulator sickness might be explained by the fact that the familiar real world remains visible in AR. Our results are consistent with findings for the HoloLens (first generation) , that reAs described in As we found the mean task time to be significantly higher for ‘voice command’ in comparison to ‘tap’, which we assume is caused by the longer action processing time of the voice-and-gaze-based interaction. It is necessary for the person to say the command, and for the device to detect and process it, while for the gesture-based interactions, the device can make use of the real-time gesture tracking. In their study with the predecessor model (HoloLens first generation), Munsinger et al.  found thIn our model, we assume that a higher required number of attempts indicates a lower efficiency. However, especially for gesture-based AR-interaction, it could be more efficient to perform several attempts in a short period of time than to only make one attempt. On the other hand, the opposite might be the case for voice-and-gaze-based interaction in AR, based on our experience. Consequently, we did not compute a single efficiency measure from the two measures used in this study, as they could scale in different directions in terms of efficiency. Therefore, the interpretation of the calculated measures regarding the efficiency of the methods must be done cautiously. The efficiency effect of performing several gesture-attempts in a shorter period appeared to be evident for the ‘air-tap’-interaction, which is the more complex gesture-based AR-interaction mode in this study. This effect might even be more prominent in more complex gesture-based interactions like the rotation of an object. However, the measure ‘mean time’ appears to be overall applicable for interpretation concerning efficiency, as a lower completion time means higher efficiency for any AR-interaction mode.We found that the children’s favorite mode to interact in AR was the ‘voice command’. However, ‘tap’ and ‘air-tap’ were chosen equally often. We therefore conclude that the preferences concerning AR-interaction vary among the children. For ‘tap’ and ‘voice command’, the most given reason for the stated preference was the perceived simplicity of the task. This indicates that the children made their choice based on their personal experience with the respective AR-interaction modes. Yet, it appears that they did not necessarily make their choice based on the efficiency measures that we applied to compare the AR-interaction modes since the ‘voice-command’-interaction was chosen most often It could be the case that the children who favored the ‘voice-command’-interaction found it easier to say the command than to tap at the object, despite performing ‘more efficient’ with the ‘tap’-interaction. The children that favored the ‘air-tap’-interaction mostly argued that it was fun to use. It appears that this choice was not made based on efficiency, but rather on personal enjoyment. However, the ‘air-tap’-interaction appears to be the least favored in general as almost 60 percent of the children claimed to not like it because of its difficulty to perform. This is consistent with our conclusion that it is the least efficient mean to interact in AR.CATML [With the aim of evaluating the applicability of the HoloLens 2 for a target group of children in educational settings, we also analyzed the influence of the HMD-AR-device on activity- related achievement emotions. Results are consistent with prior research  and indiCATML ), emotioCATML . Thus, wDue to the necessity to make individual appointments in a laboratory at a university, the sample could have a high proportion of children who are rather interested in AR  as they participated in the study on a voluntary basis and were not invited as a school class. The likely very high proportion of children in the sample with a strong interest in AR could reduce the transferability of the results to the overall population. Therefore, further research with other samples is generally needed to validate or complement the results.Moreover, the order of the AR-interaction mode presentation in the tutorial was not randomized. The lack of randomization may have produced carry-over effects from the first measuring point (‘tap’) to the second (‘air-tap’) and to the third (‘voice command’). However, this carry-over could have been diminished by the difference in execution between the presented AR-interaction modes, thus not offering many possibilities to learn something from a task that could be used when performing the next. In an experimental design with split groups for each interaction mode however, we would not have been able to assess the children’s personal interaction preference in AR in such semantic proximity to the actual tasks that they experienced as in the within-design that we chose.Due to the lack of an underlying learning content in the study, the findings cannot be directly transferred into educational situations, and they should be validated or complemented in further studies using multiple activities and learning contents as examples. Nevertheless, the present results can serve as a general basis for the development of any educational HMD-AR application.Concerning the lack of technical adaption of the device to the physical body characteristics of young children , we found that although the device was not purposely developed for children, it proved to be usable. In our study, none of the children reported problems seeing virtual objects .Research findings pointing to the general benefits of AR in educational contexts, and the present study demonstrating that HMD-AR has the potential to be used with younger learners, suggest that in the future, HMD-AR could be profitably used in classrooms. However, from an economic point of view, smart glasses are very expensive devices and if they are purchased in large numbers, e.g., for a complete course, they represent a high financial outlay for schools. In addition, there may be high software development costs, which are necessary because there are still hardly any purchasable software products for curricular-relevant contents. Therefore, schools and other educational institutions are well advised to implement AR in the classroom only if it has been proven that a specific learning content is better conveyed through AR than via traditional or less expensive media and methods. For learning content in which manual interaction with real or virtual objects is not central to learning success, one can also fall back on the more affordable tablet variant.Our study indicates a good system usability of Microsoft’s HoloLens 2 when used by elementary school children. The newly implemented direct ‘tap’-interaction in AR appears to be most effective without prior training. Despite requiring more time to perform, the ‘voice command’-interaction was found to work well with children’s voices. This deviates from previous findings for the HoloLens (first generation) and other technologies. Further, we found different interaction preferences in AR among the children in accordance with prior research. Yet, the children’s preferences do not seem to be based on objective efficiency. Our study suggests that the HoloLens 2 (as an HMD-AR-device) is generally effective for applications with young children as a target group. However, the provided AR-interaction modes appear to differ in their efficiency, at least during the time of familiarization with the device. We propose that future HMD-AR applications for education offer multiple interaction modes to serve the different interaction preferences in HMD among the children.Although the research results presented are not based on a specific learning content, they still provide an important point of reference for developers designing HMD-AR based learning applications. However, the particular integration of HMD-AR in educational situations has to be aligned with applied instructional methods and current learning goals. Prior research on HMD-VR-based lessons showed that learning outcomes vary depending on the use of a learning strategy . Neverth


Speckle-type POZ protein (SPOP), a Cullin 3-based ubiquitin ligase (CUL3SPOP), acts as a prostate-specific tumor suppressor. Loss-of-function mutations in SPOP occur in 10% of primary prostate cancer with a high Gleason grade and poor prognosis. However, it is unclear how the ubiquitin ligase activity of SPOP is controlled and how dysregulation of SPOP contributes to malignant transformation. Here, we identified GTPase Activating Protein (SH3 Domain) Binding Protein 1 (G3BP1) as an interactor and upstream regulator of CUL3SPOP, and it functions as an inhibitor of CUL3SPOP ubiquitin ligase, suggesting a distinctive mode of CUL3SPOP inactivation that aggravates prostate cancer. Speckle-type POZ protein (SPOP) is a tumor suppressor of PCa and functions as a substrate receptor of the Cullin 3 (CUL3)-based ubiquitin ligase.1–4 CUL3SPOP directs ubiquitin-proteasomal degradation of key regulators, e.g., DEK Proto-Oncogene (DEK), Bromodomain Containing 4 (BRD4), Programmed Cell Death Protein 1 (PD1) including AR, and AR co-activators Nuclear Receptor Coactivator 3 (SRC-3) and Tripartite Motif Containing 24 (TRIM24)6of the AR signaling pathway. However, the exact mechanisms of SPOP function and regulation in PCa progression remain unclear. Recently, in Nature communications, we have demonstrated a new oncogenic role of stress responsive protein GTPase Activating Protein (SH3 Domain) Binding Protein 1 (G3BP1) that negatively regulates tumor suppressive SPOP ubiquitin ligase, leading to upregulation of AR signaling and prostate tumorigenesis7 (high prostate cancer.Dysregulation of Androgen Receptor (AR) signaling is the major pathway associated with prostate cancer (PCa) progression, and agents targeting AR represent the mainstay of pharmacologic therapy for PCa. genesis7 . The stuSPOP). To understand the biochemical consequences of G3BP1 and SPOP interaction, we conducted ubiquitination assay where we observed that ectopic expression of G3BP1 reduced CUL3SPOP-dependent ubiquitination of AR, a bona fide SPOP substrate. To further understand the molecular mechanism of G3BP1-mediated SPOP suppression, we performed in vitro ubiquitination assay with affinity-purified recombinant E1, UBCH5 (as E2), CUL3/RBX1, SPOP, and DEK (substrate) and observed that G3BP1 inhibits CUL3SPOP-dependent ubiquitination of DEK in a dose-dependent manner. Moreover, using in vitro binding assay with affinity-purified proteins, we further observed that increasing doses of G3BP1 directly competed with DEK for binding to SPOP, suggesting that G3BP1 functions by excluding SPOP substrates resulting in reduced ubiquitination. Thus, it is conceivable that binding of G3BP1 to CUL3SPOP precludes substrate binding and subsequent transfer of ubiquitin to substrates, resulting in increased accumulation of SPOP substrates (such as AR TRIM24 and SRC3) that aggravate PCa progression  for SPOP interactors and identified that G3BP1 binds to and interacts with the SPOP-CUL3 complex  of 153 independent PCa cases collected at the Weill Cornell Pathology tumor bank. We observed that G3BP1 expression gradually increased in tissue samples of benign to primary PCa tumors and was most abundant in Castration-resistance prostate cancer (CRPC), suggesting that G3BP1 overexpression is associated with more aggressive disease across the clinical spectrum of prostate cancer. Interestingly, when we evaluated the probability of metastasis-free survival of primary 1,626 PCa patients stratified based on low or high G3BP1 group using genome-wide microarray gene expression data from a clinically available prognostic assay ,9 we found that patients with ‘‘high’’ G3BP1 expression showed a lesser chance of metastasis-free survival. These findings demonstrated that increased accumulation of G3BP1 corelates with tumor aggressiveness and poor metastasis-free survival.To evaluate the expression and contributions of G3BP1 in PCa patient tissue, we analyzed TCGA RNA sequencing (RNA-seq) data from 498 patient samples and observed that G3BP1 expression is high in higher prostate cancer Grade Group.10 which was found to be decreased in G3BP1-knockout cells and restored upon SPOP knockdown. This was further validated by RT-qPCR using AR target genes. Altogether, these findings suggest that G3BP1 is an upstream regulator of SPOP, and G3BP1-SPOP ubiquitin signaling axis regulates AR threshold levels and transcriptional program. To validate that G3BP1-SPOP axis mediates AR-driven signaling, G3BP1 was overexpressed in wild type primary mPECs  and AR-KO (AR knock out) mPECs. Here, we observed that G3BP1 overexpression (G3BP1OE) significantly (p < .001) enhanced organoid formation in WT mPECs, but not AR-KO and AR-KO G3BP1OE mPECs, which establishes a role for AR in G3BP1-SPOP ubiquitin signaling. To understand the clinical implication, we analyzed three PCa data sets and consistently observed that G3BP1 expression directly correlated with AR signaling both at RNA and protein levels. Moreover, we observed that G3BP1 is a bona fide target of AR that upregulates G3BP1 transcription and sets up a feed-forward loop of AR-mediated adverse signaling that further exacerbates PCa progression. These results provide compelling evidence that AR plays a significant role in G3BP1-SPOP axis-mediated downstream signaling in prostate epithelial cells.To provide further insight into G3BP1-mediated deregulation of cellular signaling pathways, we performed RNA-seq of 22RV1 cells after G3BP1 knockout or transient SPOP knockdown and compared them with control cells to identify changes in transcriptional programs. AR signaling output score was determined using a previously defined AR target gene set,high constitutes a distinct subclass of prostate cancer. G3BP1 may serve as a new prognostic biomarker in prostate cancer and provide an opportunity for precision therapy of such G3BP1high patients  mPECs significantly reduced organoid formation. Furthermore, G3BP1 knockout significantly attenuated 22RV1 xenograft tumor formation and growth. Therefore, as demonstrated herein, G3BP1patients .Figure