Conversely, plasma cell and M2 macrophage levels were lower. The follicular helper T cells in tumor cell-enriched regions were negatively correlated with resting NK cells and positively correlated with activated NK cells. In immune cell-enriched regions, this relationship was reversed. We also explored the heterogeneity of HLA gene families, immune checkpoints, and metabolism-related genes in the three regions. In tumor cell-enriched regions, we obtained 19 prognosis-related metabolism genes via univariate cox analysis. We used multiplex immunofluorescence to verify the elevated expression of SLC8A1 and MDH1 in immune cell-enriched regions and tumor cell-enriched regions, respectively, both of which were associated with prognosis of NPC. In conclusion, we explored the spatial heterogeneity of the NPC tumor environment and found specific diagnostic and prognostic markers that can be used to differentiate tumor cell-enriched regions from immune cell-enriched regions in NPC.The heterogeneity of nasopharyngeal carcinoma (NPC) leads to mixed clinical outcomes. We collected 92 regions of interest from 41 biopsies of patients with untreated NPC and obtained their transcripts using GeoMx Digital Spatial Profiling (DSP) technology. Spatial heterogeneity was determined by measuring the expression of marker genes in tumor cell-enriched (PanCK-expressing), immune cell-enriched (CD45-expressing), and normal epithelial (Endo) regions. We screened 16 prognostic markers in tumor cell-enriched regions and 4 prognostic markers in immune cell-enriched regions. The levels of CD8 Nasopharyngeal carcinoma (NPC) is mainly prevalent in North Africa, the Middle East, and Southeast Asia. Its occurrence is mainly attributed to the local environment, genetics, and infection with Epstein-Barr virus (EBV) The tumor microenvironment is composed of tumor cells, immune cells, stromal cells, metabolites, cytokines, and the extracellular matrix The current study explored the spatial heterogeneity of NPCs in both composition and function. The digital spatial profiling (DSP) technology 2 tissue cores obtained from 58 patients using a TMArrayer (Pathology Devices). Regions consisting of high tumor content with large numbers of tumor infiltrating lymphocytes (TILS) were selected following preparation of the TMA block using H&E sections from the whole tissue block to ensure the presence of NPC cells in each sample. Subsequent DSP RNA assays were performed using 41 cores including 95 regions of interest . Three regions of interest (ROIs) were excluded from paired analyses due to loss of tissue integrity during staining. Finally, 38 cores containing paired tumor cell- and immune cell-enriched ROIs were included in the differential analysis.We collected paraffin-embedded tumor tissue from 58 NPC patients who underwent initial nasopharyngeal biopsy without treatment at the Affiliated Houjie Hospital of Guangdong Medical University. This study was approved by the Ethics and Scientific Committee of the Affiliated Houjie Hospital of Guangdong Medical University. A tissue microarray (TMA) was constructed with 1.5 mmAnother commercial NPC TAM  containing 126 cases of NPC tissue were used for Immunofluorescence analysis. Five cores were excluded from analyses due to loss of tissue integrity during staining. Extreme values  were excluded from survival analysis.NanoString GeoMx DSP RNA assays were performed at CapitalBio Technology  using the standard protocol. Slides were prepared following the Manual RNA Slide Preparation Protocol in the GeoMx DSP Slide Preparation User Manual . The Whole Transcriptome Atlas (WTA) probe reagent was added to slide. We stained the tissue with GeoMx Solid Tumor TME Morphology Kit  to distinguish various morphology: epithelial cells were positively stained with PanCK while immune cells were positively stained with CD45, and nuclear stained with SYTO13. The pathologist distinguishes normal epithelial cells from cancerous epithelial cells based on histological morphology. Regions of interest (ROIs) were selected and assessed by a pathologist and illuminated using UV light. The indexing oligonucleotides released from each ROI were collected and deposited into designated wells on a microtiter plate. DSP assay sequencing data were processed with the GeoMx NGS Pipeline (DND). After sequencing, the reads were trimmed, merged, and aligned to a list of indexing oligos to identify the source probe. The unique molecular identifier (UMI) region of each read was used to remove PCR duplicates and duplicate reads, thus converting reads into digital counts. The limit of quantitation (LOQ) was estimated as the geometric mean of the negative control probes plus two geometric standard deviations of the negative control probes. Targets that consistently fell below the LOQ were removed, and the datasets were normalized via upper quartile (Q3) normalization. We used the prcomp function to perform principal component analysis from the gene expression matrix and plotted it with the scatterplot3d package.Comparisons between the two groups were performed using the Mann-Whitney U test or Wilcoxon signed-ranks test. Genes of significance were defined based on a fold change > 1.5 and p-values <0.05. Gene ontology (GO) enrichment and KEGG enrichment analyses of DEGs were performed using clusterProfiler R-packages with Benjamini-Hochberg multiple testing adjustment.2 =0.89) for the Pearson correlation coefficient matrix. A topology overlap matrix (TOM) was constructed to perform hierarchical clustering with at least 50 genes per module (minClusterSize =50). The cutreeDynamic function was used to automatically cut the clustered modules and merge similar modules (abline = 0.25). The turquoise module contained 569 genes. The green module carried 267 genes and the blue module included 1010 genes. The genes of modules were extracted for GO (q =0.05) and KEGG (p =0.05) functional enrichment analysis. The R packages involved in these analyses were: clusterProfiler, org.Hs.eg.db, enrichplot, and ggplot2. Genes from the turquoise module (n=569), green module (n=267) and blue module (n=1010) were respectively submitted to the STRING database (http://www.string-db.org/). Parameter settings were as follows: network type (full STRING network), meaning of network edges (confidence), and minimum required interaction score (high confidence (0.700). Protein interaction data obtained from the STRING database were entered into the Cytoscape software. The cytoHubba plugin was used to screen core genes in the PPI network.Weighted correlation network analysis (WGCNA) of 2940 differential genes filtered from 18675 genes from matched 38 PanCK-expressing and 38 CD45-expressing regions was performed using the WGCNA and limma packages. The gene expression matrix was converted into a Pearson correlation coefficient matrix. The adjacency matrix was constructed based on the optimal power value  obtained from 38 PanCK-expressing regions was screened for 16 prognosis-related genes by univariate Cox analysis. The optimal cutoff value of each of these genes was determined according to the surv_cutpoint and surv_categorize functions. The expression of each gene was grouped according to the optimal cutoff value, followed by survival analysis with the log-rank test. The turquoise module (n=569) of 38 CD45-expressing regions was screened for four prognosis-related genes by univariate Cox analysis. The optimal cutoff value for each of these four genes was determined according to the surv_cutpoint and surv_categorize functions. The expression of each gene was categorized according to the optimal cutoff value, followed by survival analysis with the log-rank test. The R packages involved in the above process were survival and survminer.The relative content of 22 immune cells was calculated for each of the 92 ROIs  using the CIBERSORT algorithm. Immune cells were visualized in 90 ROIs  at P < 0.05. Differences in immune cell levels among the three regions  were identified using the Kruskal-Wallis test with the reshape2 and ggpubr packages. A set of genes related to immune function was obtained based on previous studies Differential expression of HLA genes in the three regions (92 ROIs) was determined using the limma, reshape2, ggplot2, and ggpubr packages via Kruskal-Wallis test. Differential expression of immune checkpoint-related genes in the three regions was explored using limma, ggplot2 and ggpubr packages via Wilcoxon test. Correlation analysis between immune checkpoint gene expression and immune cell levels was performed in PanCK- and CD45-expressing regions using the limma, reshape2, tidyverse, and ggplot2 packages with Spearman correlation coefficients. This analysis was visualized.http://www.gsea-msigdb.org/gsea/downloads.jsp). GSVA of metabolism-related genes was performed in 92 ROIs using the packages GSEABase, GSVA, limma, and pheatmap. Parameters were set to P < 0.05 and only 20 pathways were shown. A total of 134 genes were obtained by intersecting 944 metabolic genes with 2940 differential genes (PanCK- vs CD45-expressing regions) and plotting the Venn diagram based on the Bioinformatics (http://bioinformatics.psb.ugent.be/webtools/Venn/) website. Univariate Cox analysis (P < 0.05) of 134 genes was performed in 38 (PanCK-expressing) ROIs to screen for prognosis-related metabolic genes, survival analysis, and ROC curve plotting. A total of 171 genes were obtained by intersecting 944 metabolic genes with 2514 differential genes (PanCK-expressing vs Endo regions). Univariate cox analysis, survival analysis, and ROC curve plotting of 171 genes were performed in 38 (PanCK-expressing) ROIs. The R packages involved in univariate cox analysis, survival analysis, and ROC curve plotting were survivor, survminer, and timeROC.We obtained 944 genes related to metabolism based on the literature TMA section was deparaffinized to retrieve the antigen and block endogenous peroxidase, according to manufacturer's instructions. Tissue sections were blocked with 3% BSA in TBST for 30 min, and then incubated with the antibody for CD45  for 30 min. The antibody was detected using the corresponding secondary antibody tagged with HRP, before visualization using CY3-TSA. Subsequently, antigen was retrieved again to prepare the slides for the next antibody. All samples were stained sequentially with CK  visualized with FITC-TSA, MDH1  visualized with Opal 647 TSA, and SLC8A1  visualized with Opal 594 TSA. Slides were counterstained with DAPI for nuclei visualization for 10 min and coverslipped using the antifade mountant. All markers stained with multiplex immunofluorescence were reviewed by a pathologist.Single-cell transcriptome data (GSE162025) from 10 NPC tissues with 82,622 cells was analyzed using Seurat to deal with the expression matrix. By setting filtering parameters, we filtered out cells with mitochondrial gene ratio>10% to exclude cells in abnormal state, and filtered out cells with gene number<500 or >3000 to exclude data with poor sequencing quality and non-single cells, and finally obtained 75710 cells. We used the Harmony package for data integration and batch removal. We performed unsupervised clustering using the default parameters in the Seurat package and then annotated these cell populations. We used the FindAllMarkers function in the Seurat package to calculate the differential genes between cell populations (log2FC=0.25 and P<0.05). The obtained differential genes were sorted to get the top ten highly expressed genes with significant differences for each cell population, and the expression matrix of these genes was obtained using the FetchData function, and plotted using the corrplot package.All immunofluorescence slides were scanned using the Pannoramic Digital Slide Scanner (3DHistech) and images visualized in CaseViewer2.4 (3DHistech). TMA core images were analyzed in HALO (Indica Labs). Area Quantification FL V2.1 module in Halo V3.0.311.314 analysis software was used to quantify the positive area and colocalization-positive area of the target region, respectively. Extreme values  were excluded to minimize data variability.The study protocol is shown in Figure CDK1, CCNB1, MCM2, TOP2A, CDC6, DTL, TPX2, UBE2C, KIF2C, RFC4, TYMS, MCM4, NUSAP1, MCM7, CENPF, FOXM1, RRM1, PCNA, MRPL13 and MRPL12 as the core genes were used to construct the PPI network. Turquoise modular genes including CD4, CD8A, CD3E, CD247, ZAP70, LCK, CD3G, CD3D, FYN, VAV1, LCP2, CD28, ITK, PTPRC, LAT, CD2, CD80, PRKCQ, IL2RB and CTLA4 as the core genes were used to construct the PPI network , while the turquoise module was significantly positively correlated with CD45 expression (r=0.73 and P= 8e-14) Fig. A. A tota network C. EnrichCD27, CEP85L, DOK3, MAST4, SEC24A and UPK3B appeared to serve as protection factors. Conversely, the genes BZW2, DLL4, GTPBP4, LSM4, MBD3, PAICS, PALM2AKAP2, PAQR4, RUVBL1 and TGS1 represent risk factors; the higher the expression of these genes, the higher the risk of death in patients with NPC , CEP85L (P < 0.001), DOK3 (P <0.001), MAST4 (P<0.001), SEC24A (P <0.001), and UPK3B (P <0.001) in PanCK-expressing regions of NPC patients correlated with better prognosis. In contrast, the expression of BZW2 (P <0.001), DLL4 (P <0.001), GTPBP4 (P <0.001), LSM4 (P <0.001), MBD3 (P <0.001), PAICS (P <0.001), PALM2AKAP2 (P =0.001), PAQR4 (P <0.001), RUVBL1 (P =0.006) and TGS1 (P =0.124), correlated positively with worse prognosis in patients with NPC , CCL21 (P =0.012), FCGR2C (P <0.001), and SLC8A1 (P =0.003) was associated with poorer prognosis in patients with NPC . In PanCK-expressing regions, the genes NPC Fig. . A total+ T cells, follicular helper T cells, activated NK cells, monocytes, M0 macrophages, M2 macrophages, resting mast cells, and activated mast cells differed among the three groups . The relative levels of plasma cells were the highest in CD45-expressing regions, while the number of CD8+ T, follicular helper T cells, and activated NK cells were the highest in PanCK-expressing regions. M0 macrophages were the highest in PanCK-expressing regions, while M2 macrophages were the highest in CD45-expressing regions , and activated dendritic cells with CD40 (P <0.001) and IDO1 (P <0.01). Conversely, negative correlations were found between CD4 memory activated T cells and BTLA (P <0.01), activated mast cells and CD244 (P <0.01), and CD40LG (P <0.01), macrophages M1 and LAIR1 (P <0.01)  and TNFSF14 (P <0.001), macrophages M1 and CTLA4 (P <0.001), and B memory cells and CD40 (P <0.001). Negative correlations were found between CD8+ T cells and BTNL2 (P <0.01), plasma cells with TNFSF14 (P <0.001), resting NK cells with IDO1 (P <0.01), macrophages M0 and TNFSF4 (P <0.01), resting dendritic cells and CD27 (P <0.01), and B memory cells and CD276 (P <0.01). We performed cluster annotation of 75710 cells from 10 NPC tissues and obtained a total of 18 cell populations  are shown in Figure CD200 (P =0.04), CD40 (P =0.036) and ICOSLG (P =1.4e-05) was higher in PanCK-expressing regions than in CD45. The expression of CD244 (P =0.0032), CD160 (P =0.0075), CD80 (P =8.5e-06), CD48 (P =1.3e-07), CD40LG (P =1.4e-06), CD28 (P =0.0034), CD27 (P =2.1e-09), BTLA (P =0.00033), ICOS (P =0.00075), HAVCR2 (P =0.0044), CTLA4 (P =2e-06), NRP1 (P =1.4e-09), LAIR1 (P =1e-05), LAG3 (P =0.0054), PDCD1LG2 (P =0.0016), TNFRSF8 (P =0.017), TNFRSF4 (P =0.021), TMIGD2 (P =0.019), TIGIT (P =8.2e-05), and TNFSF18 (P =0.0033) was higher in CD45-expressing regions than in PanCK-expressing regions. In Endo regions, the expression of CD200 (P =0.0013), CD70 (P =0.00057), CD44 (P =0.029), CD40 (P =0.0062), CD276 (P =0.0043), ICOSLG (P =1.1e-05), TNFRSF9 (P =0.0016), and TNFSF4 (P =0.0038) was lower than in PanCK-expressing regions, while the expression of NRP1 (P =0.046), IDO1 (P =0.0056), and VTCN1 (P =0.0025) was higher than in PanCK-expressing regions (The expression of HLA genes differed in the three regions (P <0.05) Fig. A. The ex0 P =0.03 and ICOSIn PanCK-expressing regions, metabolic genes Figure A were ma-expressing regions revealed 11 prognosis-related metabolic genes. The expression of COMT (P = 0.012), DUT (P =0.040), GMPS (P =0.043), MDH1 (P = 0.039), MDH2 (P = 0.020), NME1 (P =0.029), PAICS (P =0.011), POLR2I (P =0.039), PTGS2 (P =0.034) and UCK2 (P =0.029) increased the risk of NPC, while PLCB2 (P = 0.048) was a protective factor , GMPS (P <0.001), MDH1 (P =0.001), MDH2 (P <0.001), NME1 (P <0.001), PAICS (P <0.001), POLR2I (P <0.001), and UCK2 (P =0.004), the worse was the prognosis of NPC. In contrast, a higher expression of PLCB2 (P <0.001) was associated with a better prognosis for survival in patients with in NPC  predicted 2-, 4-, and 5-year survival rates in patients with NPC with a high degree of accuracy. MDH1 expression is higher in PanCK than in CD45 (P < 0.001). Multiplexed immunofluorescence analysis revealed higher levels of MDH1 protein in PanCK than in CD45 (P < 0.001), and the higher the MDH1 protein level (P = 0.048), the worse was the prognosis of patients with NPC  Fig. B. Univartor Fig. C. In PanNPC Fig. D. The RONPC Fig. F.ADCY3 (P = 0.027), ADCY9 (P =0.028), CYP2J2 (P = 0.038), DNMT1 (P = 0.024), EPHX1 (P =0.022), MBOAT7 (P =0.018), POLD1 (P = 0.016) and UAP1 (P = 0.041) correlated positively with survival risk in patients with NPC , and were also metabolism-related genes. The expression of these genes correlated with a higher risk of death in patients with NPC.Higher expression of ADCY3 (P = 0.005), ADCY9 (P =0.008), CYP2J2 (P = 0.001), DNMT1 (P <0.001), EPHX1 (P <0.001), MBOAT7 (P <0.001), POLD1 (P = 0.001) and UAP1 (P < 0.001) was associated with worse prognosis in patients with NPC  B. Univarwith NPC C. Notablwith NPC D. The ROEither bulk-RNA seq or microarray sequencing was used to explore biomarkers of NPC before GeoMx DSP technology emerged CEP85L, KRT5, MAST4, MYO1G, SLA, SMARCC2, UPK3B, ATF5, BEX3, BIK, CADM4, CDK2AP1, CLDN1, DLL4, IGFBP2, NFE2L3, PTGS2, SMC1A, SUSD4 and VRK2) and 4 prognostic markers in immune cell-enriched regions . We also screened for metabolism-related genes that affect the prognosis of patients with NPC in tumor cell-enriched regions .Using DSP technology, we screened 20 prognostic gene markers located in tumor cell-enriched regions  associated with PDAC cell survival + T, CD4+ T-helper, naïve T-cells, cytotoxic T-cells, exhausted T-cells, and Tregs) and B cells + and CD8+ T cell clusters in NPC were highly activated and depleted and co-expressed effector markers such as IL-2, GZMB, INFG, NKG7, GNLY, and GZMK. They also expressed depletion markers such as PDCD1, HAVCR2, LAG3, TIGIT, and CTLA4 + T, follicular helper T cells, activated NK cells, and M0 macrophages were higher in tumor cell-enriched regions than in immune cell-enriched regions. In contrast, plasma cells and M2 macrophages were more abundant in immune cell-enriched regions, suggesting that cellular immunity was predominantly exerted in tumor cell-enriched regions, while humoral immunity was mainly observed in the peritumor area. Interestingly, in tumor cell-enriched regions, follicular helper T cells were negatively correlated with resting NK cells and positively correlated with activated NK cells. In immune cell-enriched regions, the opposite was true, highlighting the need to use single-cell sequencing to further explore the interactions between these two cell types. In tumor cell-enriched regions, PDCD1 expression was negatively correlated with the levels of monocytes and CD4+ memory-activated T cells. The expression of LAG3 positively correlated with the levels of CD4+ memory-activated T cells and CD8 T cells. The expression of TIGIT positively correlated with monocyte levels and negatively correlated with follicular helper T cell levels. In immune cell-enriched regions, the PDCD1 expression negatively correlated with the number of eosinophils and CD4+ naïve T cells. The expression of HAVCR2 negatively correlated with the number of gamma/delta T cells and positively correlated with the levels of CD4+ memory resting T cells. The expression of LAG3 positively correlated with the levels of CD4+ memory activated T cells, CD4+ memory resting T cells, M1 macrophages, and CD8+ T cells, but negatively correlated with the number of plasma cells. The expression of TIGIT was positively related with the levels of Tregs, CD8+ T cells, and CD4+ memory resting T cells, while negatively correlated with the number of plasma cells. The expression of CTLA4 positively correlated with the levels of CD8+ T cells, CD4+ memory resting T cells, activated NK cells, and M1 macrophages, while negatively correlated with the number of naive B cells. We therefore analyzed the relationship between these immune cells and depletion markers in locations that were complementary to previous studies. However, the specific mechanisms of interactions between cells at various spatial locations need to be further explored.The predominantly infiltrating lymphocytes in the NPC microenvironment are T (CD8Limitations of this study include the small sample size, which restricts the correlation between the tumor immune characteristics, treatment, and outcomes. Even though our study is small the strength is the paired samples. Another limitation is that the biomarkers we screened have not yet been validated in functional experiments, and the roles of these biomarkers still need to be further elucidated through in vivo and in vitro experiments in the future.In conclusion, we used DSP technology to explore the heterogeneity of the NPC microenvironment and its regional and metabolic prognostic markers and characterized the immune infiltration. The screened prognostic biomarkers may provide a theoretical basis for clinical diagnosis, targeted therapy, and new drug development.Supplementary figures and tables.Click here for additional data file.


Metastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. in vitro and in a metastatic xenograft mouse model.Here, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors.Our results show that NK-92/5.28.z cells effectively kill RMS cells These findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS. AdoleDeveloping new therapeutic strategies to overcome tumor resistance remains a critical unmet need for these heavily pretreated patients. Chimeric antigen receptor (CAR)-engineered cells, which target and inhibit the proliferation and spread of tumor cells, are revolutionizing the treatment of r/r malignancies. However, this therapy can also lead to severe, albeit treatable, side effects, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and secondary hemophagocytic lymphohistiocytosis/macrophage activation syndrome .The lack of suitable CAR target antigens, as well as the immunosuppressive nature of the tumor microenvironment (TME), and advanced-stage disease, renders CAR-T cells nonresponsive or exhausted against many solid tumors , 9. HoweHere, we employed a molecularly and functionally well-defined clonal derivative of the natural killer (NK) cell line NK-92 (NK-92/5.28.z) as effector cells –17, carrin vitro  aRMS xenograft model.To further develop the NK-92/5.28.z cell therapy towards clinical application as a consolidation treatment for children and young adults with r/r, metastatic, ErbB2-positive RMS tumors after re-induction radiotherapy/chemotherapy, we performed detailed preclinical 22.1The NK-92 cell line was derived from a 50-year-old patient with non-Hodgkin´s lymphoma . NK-92/5PAX3-FOXO1) derived from a patient with alveolar RMS was purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with GlutaMAX (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) . For in vivo cell tracking, stable GFP/luciferase-expressing RH30 cells (RH30GFP/luc+) were generated by lentiviral transduction with the pSIEW-luc2 plasmid encoding enhanced green fluorescent protein (eGFP) and firefly luciferase linked by a T2A peptide (GFP/luc+ cells were enriched by fluorescence-activated cell sorting using a FACSAria II instrument (BD Biosciences).The aRMS cell line RH30 ( peptide . GFP-posPAX3-FOXO1), RMS127 (PAX3-FOXO1), and RMS335 (PAX7-FOXO1) were generated from tumor biopsies of patients  and subsequently stained with ErbB2 antibody for 20 minutes. After washing, 1x104 events were recorded by flow cytometry using FACSDiva software . Fluorescence minus one (FMO) controls were used as references. The actual ErbB2 receptor number was quantified using BD Quantibrite Beads (BD Biosciences) according to the manufacturer’s instructions. ErbB2 expression was determined using FlowJo Software .Tumor organoid RMS cells were stained with phycoerythrin (PE)-conjugated antibody  for ErbB2 and subsequently analyzed with a FACSCanto 10c flow cytometer (BD Biosciences). Cells were stained according to the manufacturer’s instructions. In brief, aliquots of 5x102.3The short-term toxicity of NK-92/5.28.z cells against tumor organoid RMS cells was determined using a europium release assay as reported elsewhere . In brie2.45 cells were washed in phosphate-buffered saline (PBS) and subsequently stained with ErbB2 antibody or an IgG1κ isotype control  for 20 minutes according to the manufacturer’s instructions. After washing, 1x104 events were recorded by flow cytometry using FACSDiva software . In addition, ErbB2-CAR surface expression of NK-92/5.28.z cells was assessed. For this purpose 2x106 either NK-92/5.28.z or NK-92 cells were washed once with DPBS and then incubated with Human TruStain FcX™ (Biolegend) for 20 minutes at 4°C. After another washing step in DPBS, the cells were incubated with 10 µg recombinant ErbB2 with human Fc tag for 20 minutes. After two additional washing steps, cells were stained with APC antibody directed against the Fc tag (Biolegend), 410712 cells, washed again, and analyzed using a BD FACS Canto 10c instrument (BD Biosciences).The integrity of RH30 target and NK-92/5.28.z effector cells was verified. In brief, aliquots of 5x10in vitro: tumor cells were resuspended and were counted by Neubauer-improved hemocytometer. 5000 RH30GFP/luc+ cells per well were seeded in 200 μl of RPMI +10% FBS into ultralow attachment 96-well round-bottom plates without prior coating (Corning) and spun down. On day four of culture, 100 µl of supernatant was carefully removed, and 1x105 NK-92 or NK-92/5.28.z cells resuspended in 100 µl of NK-92 medium were added. Spheroids without effector cells were used as controls. Every three to four days, the medium was replaced by X-Vivo 10 medium with 5% FFP and 100 IU/ml IL-2. Spheroids were imaged on day 4, 5, 6, 8 and 10 after initial seeding using a Celigo Image Cytometer (Nexcelom Bioscience) with F-theta lens and AVT PIKE camera. The size of spheroids was quantified by the GFP signal using Fiji software (Version 2.3.0)  and were conducted according to the requirements of the German Animal Welfare Act.5 RH30GFP/luc+ cells resuspended in 100 µl of PBS were injected intravenously (iv) via the tail vein. Considering them as having an immanence risk for disease progression with then limited treatment options as previously shown  or NK-92/5.28.z cells . Both effector cell types were resuspended in X-Vivo 10 medium with 5% FFP and 100 IU/ml IL-2. Group size was set based on the experience from previous experiments with the same xenograft model.Twenty-seven female 10- to 12-week-old nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/Il2receptorgamma−/− (NSG) mice received sublethal irradiation with 2.5 Gy (Biobeam 2000) (d-1) according to the protocol for previously established metastatic RMS xenograft model closely resembling a clinical situation . One dayly shown , immunotin vivo grade VivoGlo luciferin (Promega) in 100 µl of PBS per mouse. Fifteen minutes later, images were acquired in dorsal and ventral positions and subsequently analyzed by Living Image In Vivo Imaging Software (Perkin Elmer). Uniform regions of interest were used for all mice, and total flux (photon/s) was used for measurement. The signals of dorsal and ventral images were combined into a luminoscore, and statistical analysis of the tumor burden was performed by one-way ANOVA (Tumor growth was monitored weekly by bioluminescence imaging (BLI) using an IVIS Lumina II system (Perkin Elmer). Mice were anesthetized by isoflurane inhalation and received subcutaneous injections of 150 µg ay ANOVA .Altogether, tumor-bearing mice were randomly divided into the following three groups:- Control: one course of six serial infusions with X-Vivo 10 media with 5% FFP and 100 IU/ml IL-2, n=56 cells, one course of six serial effector cell infusions, n=12- NK-92: 10x106 cells, one course of six serial effector cell infusions, n=10- NK-92/5.28.z: 10x10During the experiment, animals were monitored daily for disease symptoms, xenogenic graft versus host disease (GVHD) and other adverse effects of the NK cell therapies for a maximum of 105 days. Mice with visible signs of disease progression, discomfort or physical abnormalities were painlessly sacrificed by isoflurane anesthesia followed by cervical dislocation.2.5.1Peripheral blood (PB), BM, lung, liver, gut and spleen samples were isolated and analyzed for persistence of human tumor or effector cells: BM was flushed out of the femur and tibia of mice. PB and BM were incubated with red blood cell lysis buffer (RBC Lysis Buffer (10x), BioLegend) according to the manufacturer´s instructions and washed once with PBS. The organs were cut in representative halves, and one half was preserved in formaldehyde for fluorescence microscopy. The other half was incubated with collagenase D solution , filtered through a 70 µm cell strainer, and washed with PBS. Aliquots of the cell suspensions were analyzed by flow cytometry and quantitative polymerase chain reaction (qPCR). Sample identity was blinded for the executors of the consecutively analyses.2.5.2GFP/luc+ was assessed shortly before use in experiments with a FACS Canto 10c device (BD Biosciences). CAR expression of NK-92/5.28.z cells was analyzed using a chimeric ErbB2-Fc protein  after nonspecific Fc receptor blocking  and subsequent staining with an anti-IgG-Fc secondary antibody conjugated with allophycocyanin (APC) .GFP expression of RH30Cell samples from mouse organs were washed once in PBS and stained with phycoerythrin-cyanin 7 (PE/Cy7)-conjugated anti-human CD45 antibody .Analyses were performed with FlowJo Software .2.5.3Genomic deoxyribonucleic acid (DNA) from murine organs was extracted using the Extractme Genomic DNA Kit (BLIRT S.A.). The percentage of human cells in the different murine tissues was determined by a quantitative real-time approach specifically amplifying the human albumin gene. In a second step, the proportions of NK-92 or NK-92/5.28.z cells and tumor cells within the human cell fraction were determined by a human-specific short tandem repeat (STR) genotyping approach. The tumor burden of each mouse was quantified in organs previously described as RMS metastatic sites  , 32. For2.5.4For histological analyses, organ sections were fixed in 4% buffered formalin, paraffin embedded, cut, and stained with hematoxylin-eosin (HE). Immunohistochemistry (IHC) antibodies targeting CD56, MYOD1, DESMIN and MYOGENIN were used to evaluate the RMS phenotype of the tumors. The stained tissue sections were rated by a pathologist for the expression of each protein .2 fragment goat-anti-mouse conjugated to Alexa 647  and goat-anti-rabbit IgG conjugated to Alexa 488  were used, and nuclei were stained with 4´,6-eiamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The localization of immune cells in the tumor tissue relative to blood vessels was determined by staining with rat-anti-mouse Meca32 antibody  and secondary goat-anti-rat Alexa546 antibody . The samples were examined with a BZ-X810 All-in-One Fluorescence microscope (Keyence) with basic lenses  and filter cubes Ex: 360/40 DM: 400 BA: 460/50 (DAPI), Ex: 470/40 DM: 495 BA: 525/50 (GFP), Ex: 620/60 DM: 660 BA: 700/75(Cy5).Immunofluorescence staining was used to observe immune cell infiltration in organs and tumors: organ slices were deparaffinized and rehydrated. After antigen retrieval and a blocking step, primary mouse-anti targeting human CD45 antibody  and rabbit-against GFP antibody  were added. For detection, secondary antibodies F(ab`)In addition, the stained tissue sections with immune cell infiltration were analyzed by a pathologist for xenogenic GVHD.2.6p< 0.05 (*), p< 0.01 (**), p< 0.001 (***) and p<0.0001 (****) were considered statistically significant.For statistical analyses and graphical presentation, GraphPad Prism software  was used. The results are given as the mean ± standard deviation (SD). Differences between different groups were evaluated by two-tailed Student´s t test or one-way ANOVA using the Bonferroni-Dunn (nonparametric) method. Overall survivals are given as median with 95% confidence interval, Differences between the survival of different treatment groups were analyzed by the log-rank (Mantel-Cox) test. Differences with 33.1in vitro assessment of efficacy, parental NK-92 or NK-92/5.28.z cells were tested against patient-derived tumor organoid aRMS cells in a 2D europium release assay. Maintaining actively growing rare patient-derived cells is challenging, but the tumor organoid RMS cells RMS102 (PAX3-FOXO1), RMS127 (PAX3-FOXO1) and RMS335 (PAX7-FOXO1) could be successfully passaged in vitro using previously published cultivation conditions  on day 10 (p = 0.0013) and NK-92-treated spheroids (p = 0.0425)  xenograft were used to assess homing, tumor invasion, persistence, and antitumor effects as well as the xenogenic cytotoxicity (GVHD) of parental NK-92 and NK-92/5.28.z cells. Sequential infusions of effector cells were given preemptively during the low tumor burden period, and mice were followed for 105 days (end of experiment) (p = 0.0101) and NK-92-treated (p = 0.0159) animals at the time of first in vivo response assessment on day +50 (p = 0.2120) . In contrast, NK-92/5.28.z cell therapy (81.0 ± 8.8 days) significantly improved overall survival compared to that of the parental NK-92-treated (p = 0.0329) and untreated controls (p = 0.0366) . All detectable tumor lesions expressed CD56 , MYOD1 , DESMIN  and MYOGENIN .Tumor lesions were primarily observed in the livers and lungs of mice except in those with complete response to immunotherapy with NK-92/5.28.z cells. Multiple large (macroscopic) lesions were more likely to be present in the untreated and NK-92-treated mice vs. single small (microscopic) lesions in the NK-92/5.28.z-treated mice . In vivo treatment of mice with parental NK-92 cells had no significant effect, indicating that the observed therapeutic efficacy of NK-92/5.28.z cells was mainly mediated by the second-generation ErbB2-CAR construct.In summary, our data show that NK-92/5.28.z cells exhibit high cytotoxicity in r/r ErbB2-positive 2D patient-derived tumor organoid RMS cells and 3D aRMS tumor spheroid models in vivo expansion of NK-92/5.28.z cells upon irradiation may be addressed by repeated dosing and combination with checkpoint inhibition or other anticancer therapies that could further enhance or benefit from the immunomodulatory activity of the cells.NK-92/5.28.z cell therapy is already under safety evaluation in patients with recurrent glioblastoma, which is an advantage when considering to extend clinical application of this ready-to-use CAR-engineered product to other cancer indications such as metastatic aRMS. We therefore propose to use NK-92/5.28.z cells for consolidation therapy of r/r, metastatic ErbB2-positive RMS following re-induction radiotherapy/chemotherapy to benefit from their multiple endogenous cytolytic pathways in addition to direct CAR-mediated killing. Thereby, the limited life-span and lack of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was approved by Regierungspräsidium Darmstadt, Darmstadt, Germany; Gen.-Nr. TVA FK/1070. The study was conducted in accordance with the local legislation and institutional requirements.CH, LM, WW, EU, HB, MM and ER conceived and designed the experiments. CH, LM, HK, HB, EG, EU, MM and ER performed the experiments. CH, LM, HK, HB, EU, MM and ER analyzed the data. HK, HB, TT, WW, EG, EU, MM, MK, FH, JD, J-HK, PB contributed to reagents, materials and analysis tools. CH, LMM and ER wrote the manuscript. HK, HB, TT, WW, EG, EU, MM, M, FH, JD, J-HK, PB and MM revised the manuscript. J-HK and PB supervised the research. All authors contributed to the article and approved the submitted version.


Correction: Implementation Sci 18, 31 (2023)https://doi.org/10.1186/s13012-023-01286-zThe original publication  of this In the second paragraph, Lyon and Bruns was incorrectly cited as reference 1, where it should have been cited as reference 70.The correct reference 70 is supplied below:70. Lyon AR, Bruns EJ. User-Centered Redesign of Evidence-Based Psychosocial Interventions to Enhance Implementation—Hospitable Soil or Better Seeds? JAMA Psychiatry. 2019;76(1):3–4. doi:10.1001/jamapsychiatry.2018.3060The original article  has been


Ornithobacterium hominis type strain MSHR-COH1 (ATCC TSD-185/NCTC 14317), a bacterial species isolated from the human nasopharynx. Long-read sequencing reveals that the genome is 2,036,909 bp in length, with a GC content of 35.72%.We report the complete genome sequence of the  Ornithobacterium hominis is a species in the family Weeksellaceae, for which partial genomes were first described from nasopharyngeal samples from Thailand  (ATCC TSD-185), streaked on Columbia agar with 5% sheep blood , and incubated at 37°C for 72 h under humid microaerobic conditions  using the CampyGen atmosphere generation system  plus approximately 20 mL water. All bacterial colonies were scraped into phosphate-buffered saline and immediately processed using the MasterPure complete DNA/RNA purification kit  following the manufacturer’s protocol. The sequencing library was prepared with the 24-plex native barcoding kit v12 . No DNA shearing or size selection was employed.N50 value of 10,303 bp and an N90 value of 5,608 bp.The library was sequenced on an ONT MinION device with an R10.4 flow cell , with Guppy v6.4.6 base calling using the SUP model and adaptor/barcode removal. Reads were trimmed using Prowler (SRR8360295]) mapped with Minimap2 v2.24 , and 3 rRNA operons. Sites that may disrupt short-read assemblies include the rRNA operons, 450-bp noncoding sequences associated with putative Fibrobacter succinogenes domain genes, >500-bp rearrangement hotspot (RHS) domain sequences, and >1-kb sequences within putative type III fibronectin domain genes. Unusually, the dnaA gene is situated near the proposed genome terminus rather than near the origin of replication , Flye v2.9.2 (https://github.com/fenderglass/Flye), Trycycler v0.5.3 (https://github.com/rrwick/Trycycler), Ori-Finder 2022 (http://tubic.tju.edu.cn/Ori-Finder2022/public/index.php) (accessed 11 January 2023), Pilon v1.24 (https://github.com/broadinstitute/pilon), Minimap2 v2.24 (https://github.com/lh3/minimap2), and Proksee (https://proksee.ca) (accessed 27 April 2023).The following software was used: Guppy v6.4.6 (ONT), Prowler (commit ID c3041ba)  under BioProject accession number


Urinary tract infections (UTIs) are common healthcare-associated and community-acquired bacterial infections in children. Data on pediatric UTIs in the Gulf Cooperation Council (GCC) region  have not been collated. Our aim is to review the published literature on the risk factors, etiology, antimicrobial susceptibility, and treatment of pediatric (aged <18 years) UTIs from healthcare and community settings in the GCC countries. Up toEscherichia coli accounts for 80%–90% of pediatric UTIs (E. coli urinary isolates from children in the Gulf Cooperation Council (GCC) region  (Uropathogenic s (UAE)] . Furthers (UAE)] .The challenges of managing pediatric UTIs in the GCC region have not been documented in a single article. Therefore, this review will collate data on risk factors, pathogens, resistance phenotypes, and antimicrobial management practices for pediatric UTIs within the region. Our objective is to provide a comprehensive assessment of the current knowledge on pediatric UTIs to improve diagnostic accuracy and help clinicians make rational therapeutic choices for UTIs, to improve the health of children in the region.2.2.1.This review evaluated relevant studies published between 1st January 2011 and 31st March 2022 on the PubMed and Google Scholar databases. We excluded non-English language articles, case reports, letters, books, editorials, notes, and conference abstracts.The primary search terms were “urinary tract infection” and “pediatric”, followed by secondary terms relating to the review topics shown in . A free 2.2.Following initial identification and screening of publication titles, 38 full-text articles remained . Nine articles  were excluded for not differentiating data for extraction.The final GCC study number totaled 28: 24 retrospective studies –34 and 43.3.1.The Saudi Pediatric Infectious Diseases Society (SPIDS) guidelines on community-acquired UTI (CA-UTI) recommend transurethral catheterization for collecting urine from infants and non-toilet-trained children to minimize bacterial contamination from the skin . Cathete3.2.SPIDS defines UTI as significant bacteriuria in a symptomatic patient (febrile and/or with urinary symptoms) and recurrent UTI as ≥2 episodes of symptomatic UTI within 12 months . Their tP = 0.001) (P = 0.044) and nitrite (P = 0.046) were significantly sensitive to detect E. coli UTI, compared with non-E. coli UTI , 2815, 2= 0.001)  26). Py. PyP = 0coli UTI  (26).3.3.SPIDS advocates renal and bladder ultrasonography as a safe, noninvasive procedure for the detection of abnormalities and renal infections in preference to micturating or voiding cystourethrogram (MCUG or VCUG) . Renal u3.4P < 0.001)] (The predominant symptom was fever (50.3%–86.6%), with a regional prevalence of 60.7%   15, 16,, 27, 32. 0.001)]  (32). Tw 0.001)] , 35.P = 0.002), whereas rates of abdominal pain and urinary symptoms were higher in older children [2–14 years (P < 0.001)] (P < 0.001) (Other common symptoms (>20% of each study cohort) were urinary frequency, urgency or dysuria, nausea, vomiting, and abdominal pain  15, 16,,16, 25. < 0.001)  32)..P = 0.00Blood C-reactive protein (>5 mg/L) did not significantly change with patient age  or betwe4.4.1.4.1.1.The prevalence of pediatric UTI across four studies including adults was 18.6%–68.7% , 33. In 4.1.2.P < 0.001), P = 0.001)]. Boys presented with CA-UTI at a significantly younger age than girls  of UTI patients were girls. More girls than boys had a UTI in 15 studies  11, 12,, 36, 38; of UTI pan girls  (11).4.1.2.1In other studies, being uncircumcised was associated with UTI , 41. In 4.2.4.2.1.P = 0.018), antibiotic use (P = 0.015) and VUR (P = 0.019) were associated with UTI (P = 0.02) and duration of fever of >36 h (P = 0.001), but not with the isolated uropathogen ..P = 0.01P = 0.05) (P = 0.025) and age >2 months (P = 0.006) (P < 0.0001)  were associated with UTI ( = 0.05) . UTI amo= 0.006)  14). In. InP = 0 0.0001) .Following cardiac surgery, 7.0% of patients had UTIs  . Prolong4.2.2.E. coli, non-E. coli UTIs were associated with hospitalization >7 days (P = 0.042), male sex (P < 0.0001), younger age [<4 years (P = 0.01)], prior use of antibiotics (P = 0.011), or history of UTI (P = 0.012) (E. coli UTIs (E. coli species (E. coli (49%) and non-E. coli (51%) in patients with normal ultrasound were comparable (Compared with = 0.012)  28). Si. SiE. cooli UTIs  .4.2.3.E. coli, and ESBL-producing E. coli were associated with VUR in children (P = 0.044) ; in neonates than older children and infants (P = 0.016) (One study of adults and children in Saudi Arabia found that significantly more UTI patients had non-ESBL-producing than ESBL-producing = 0.044)  12). Th. ThE. co= 0.016) ; and in = 0.016) . In cont= 0.016)  22). An. AnE. co= 0.016)  and 40.64.2.4.P < 0.005)]   13, 15,, 32. The 0.005)] .5.5.1.E. coli was predominant (26.1%–100% of isolates), with a regional prevalence of 64.9%   and 19.8% (37 of 187 K. pneumoniae), respectively (P = 0.05) (thogens) , 32–34. thogens)  11, 12,thogens) . The reg,thogens ectively , 32–34.  = 0.05)  (11).Klebsiella spp. (predominantly Klebsiella pneumoniae) were the second most common uropathogens (3.8%–35.2% of isolates), with a regional prevalence for K. pneumoniae of 13.8%  (thogens) , 27, 30.Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus spp., and Enterobacter spp.  and cefazolin (94.0%) in Bahrain and Saudi Arabia  (P < 0.001) for recurrent UTIs , 34E. coi Arabia  among E. to 2020  (34). Moeropenem . In Omanent UTIs .E. coli from children in Bahrain (E. coli) from Qatar were resistant to ceftriaxone (More than 96% of ESBL-producing  Bahrain  34) and andE. cotriaxone  36)..E. coli K. pneumoniae, >90% of isolates were susceptible to cefotaxime, ceftriaxone, and meropenem  was 26.9% among enterococci  from recurrent UTIs (P < 0.001) . CoE. coli, 83%) from Qatar were resistant to amikacin, with higher resistance to nitrofurantoin (13.0%), gentamicin (24.4%), and trimethoprim-sulfamethoxazole (59.7%) (E. coli from Bahrain was highest to nitrofurantoin and fosfomycin (71.0%–100%) (No ESBL-producing isolates ( (59.7%)  36). Su. SuE. co0%–100%)  33). Al. AlE. co0%–100%) .K. pneumoniae, >90% of isolates were susceptible to the tested aminoglycosides . Th. ThE. co5.3.E. coli isolates (61.1%) harbored the blaCTX−M−G1 gene, whereas 46.2% of ESBL-producing K. pneumoniae carried blaSHV, blaTEM and blaCTX−M−G1 genes (Most pediatric ESBL-producing G1 genes  36). Th. ThE. coE. coli and K. pneumoniae (52.2% of isolates were urinary) (Similar findings were demonstrated by a study in Qatar of pediatric ESBL-producing urinary) . CTX-M gurinary) .E. coli; 30% K. pneumoniae) in Qatar revealed that carbapenem resistance was largely attributed to a limited number of OXA-48-like and NDM carbapenemases, suggesting that certain international high-risk clones associated with other carbapenemase genes such as blaKPC-type, have not spread locally , mostly a cephalosporin (43.5%) or penicillin (44.2%), namely amoxicillin-clavulanic acid (29.4%) or cefprozil (23.0%)  19). In. In19). 7.The evidence presented in this review indicates limited published data from the UAE and a lack of studies on HA-UTIs. Also, most studies specified a patient age of <14 years, while four studies included patients aged 14–18 years , 33. GCC8.E. coli (including ESBL-producing isolates). ESBL-producing uropathogens are an established cause of UTI in the region, whereas CRE organisms  are emerging  should be reserved for critically ill children, particularly those having a previous UTI caused by, or other risk factors for, ESBL-producing uropathogens. Alternatively, ceftazidime-avibactam, a novel cephalosporin/β-lactamase inhibitor combination that inactivates ESBL and OXA-48-type enzymes, could be considered a treatment option for ESBL pyelonephritis (Empirical use of aminoglycosides due to the high prevalence of uropathogens resistant to 3GC has been proposed in Kuwait . Gentamiountries , 36. Theountries , 48–51. ephritis .E. coli in our region.The treatment of UTIs caused by carbapenemase-producing uropathogens in our region is challenging. Although aztreonam is stable against NDM and 3GC against OXA-48 enzymes, none of these agents can be empirically used to treat UTI caused by NDM and OXA-48 producers as most isolates in our region co-produce CTX-M-type ESBL . It coulIn conclusion, our review showed that UTIs are increasingly caused by antimicrobial-resistant uropathogens in the pediatric population of GCC countries. Further epidemiological and clinical studies are needed to optimize diagnostic and antimicrobial stewardship strategies in pediatric UTIs in the GCC region.


Correction: Microbiome 10, 226 (2022)https://doi.org/10.1186/s40168-022-01430-9Following publication of the original article , the autThe updated additional file is included here and original article has been updated.Additional file 1:Fig. S8. L. reuteri improves metabolic control in DIO mice treated with CQA. HFD-fed mice were treated twice per week with L. reuteri + CQA  by oral gavage for 5 weeks. Related to Fig. 6. (a) GTT and AUC. (b) Serum HDL-C. (c) Serum LDL-C. (d) Liver weight. (e) Hepatic mRNA expression of lipid synthesis-related genes.  Representative FL-IR images and BAT temperature. (h) Relative mRNA expression of thermogenic genes in BAT. (i) Representative H&E (upper) and UCP1 (lower) staining of BAT sections, scale bar: 50 μm. n = 8/group. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01; and ***, p < 0.001. ns means not statistically significant.


Dicranomyia (Erostrata) jejuensissp. nov. and D. (E.) koreanasp. nov., from Korea are described on the basis of morphology and mitochondrial COI sequences. DNA barcode sequences for other four D. (Erostrata) species from Korea are also provided for the first time. The identification key for all known D. (Erostrata) species is presented.Two new crane fly species,  Dicranomyia Stephens, 1829, is the largest genus of the Limoniidae and, as such, contains 1,136 species and 24 subgenera, including the subgenus D. (Erostrata) Savchenko, 1976. Twelve species of this subgenus have been reported from the Palearctic, Nearctic, and Oriental regions (D. (E.) globithorax has been reported in fungus on decaying logs  tabashii (as Limonia (Limonia) tabashii) from Suigen (= Suwon) in Korea, and Dicranomyia species of the subgenus Erostrata including three new species and suggested that Limoniacongesta Alexander, 1976 and Limoniastriopleura Edwards, 1919 be classified as members of subgenus Erostrata based on non-genitalic characters.D. (Erostrata) crane fly species are described from Korea, providing identification for all Korean members of the subgenus. A DNA barcode (COI) dataset for six Dicranomyia species of the subgenus Erostrata from Korea is also presented for the first time.Here, two new Crane fly adults were collected using insect nets or Malaise traps and preserved in 80% ethanol Table . Wings aThe terminologies used to describe the morphology generally follow KUEM – Korea University Entomological Museum, Seoul, Republic of Korea;NIBR – National Institute of Biological Resources, Incheon, Republic of Korea.Specimen depositories are as follows:COI sequences were amplified and sequenced following COI. All sequences were submitted to GenBank .Total genomic DNA was extracted from the leg muscle of using the DNeasy Blood & Tissue Kit  according to the manufacturer’s instructions. COI sequences (Table D. (Erostrata) species (10 sequences), and the outgroup species D. (Dicranomyia) kandybinae Savchenko, 1987 (1 sequence). Phylogenetic analyses were conducted using the neighbor-joining (NJ) method and Kimura-2-parameter model (p-distances) in MEGA X, using the complete deletion option.DNA barcode analysis was performed using 11 er model , with 1,er model . SequencDicranomyia (Erostrata) canis Dicranomyia (Erostrata) cnephosa Dicranomyia (Erostrata) congesta Dicranomyia (Erostrata) cynotis Dicranomyia (Erostrata) globithorax Osten Sacken, 1869Dicranomyia (Erostrata) globulithorax Alexander, 1924Dicranomyia (Erostrata) jejuensis sp. nov.Dicranomyia (Erostrata) koreana sp. nov.Dicranomyia (Erostrata) melas Dicranomyia (Erostrata) reniformis Kato, Tachi & Gelhaus, 2018Dicranomyia (Erostrata) striopleura Dicranomyia (Erostrata) submelas Kato, Tachi & Gelhaus, 2018Dicranomyia (Erostrata) tabashii Dicranomyia (Erostrata) yazuensis Kato, Tachi & Gelhaus, 2018Limoniidae Speiser, 1909Family Limoniinae Speiser, 1909Subfamily Taxon classificationAnimaliaDipteraLimoniidaeSubgenusSavchenko, 1976C69BCB6D-4C3F-5FAE-A61B-87A541D8AD53Dicranomyia (Erostrata) Savchenko in Dicranomyiaglobithorax Osten Sacken, 1869 .Rostrum is very short or reduced. Number of palpomeres ranges from one to three. Wings have no patterns, even in stigmal region. Third and fourth tarsomere are slightly swollen. Internal sac or notch is located on the male seventh sternite. Gonocoxite has ventromesal lobe. Gonostylus is one paired, with one or two setae arising from small tubercle on outer surface.Taxon classificationAnimaliaDipteraLimoniidae15076BEE-3A44-562E-8FC1-6CF43A4FF557https://zoobank.org/9659D5F8-C578-4FAA-B16A-6AF8A9AE39E5Holotype: Korea • ♂; Jeju-do, Seogwipo-si, Namwon-eup, Sillye-ri, Iseungi-oreum Volcanic Cone; 33°20.24'N, 126°37.25'E; alt. 450 m; 4 Aug.–8 Sep. 2021; Y. J. Bae leg.; Malaise trap; GenBank: OM102981; CF21-0150H; NIBR.Paratypes: Korea • 1 ♀; same data as holotype, 14 Jul.–4 Aug. 2021; GenBank: OM102983; CF21-0151; KUEM • 1 ♂; same data as holotype; GenBank: OM102982; CF21-0150P; KUEM.Palpus is 3-segmented. Male seventh sternite has shallow V-shaped notch. Outer face of gonostylus has single seta arising from tubercle. Distal lobe of paramere has a hooked tip with a subapical process.Male (holotype). Body length 4.3 mm, wing length 4.6 mm, antenna length 0.9 mm. General body coloration pale yellow to yellowish brown . General body coloration brighter than male. Femur I 2.8 mm; II 3.2 mm; III 3.4 mm; tibia I 3.2 mm; I: 3.1 mm; III 3.4 mm; tarsus I 3.0 mm; II 2.6 mm; III 2.4 mm.Female terminalia  submelas.Adults of this species are found in deciduous forests with moss-covered rocks along intermittent, rocky mountain streams Fig.  and co-oAdults were collected from June through early September.Dicranomyia (E.) jejuensis sp. nov. is morphologically similar to D. (E.) yazuensis based on the male genital structures, but it can be distinguished by the following characters: pleuron entirely dull yellow ; palpus 3-segmented (vs 2-segmented); distal 1/2 of gonostylus tapered to tip ; posterior margin of male seventh sternite with shallow V-shaped notch (vs long triangular notch); distal part of paramere with hooked tip (vs straight tip).Taxon classificationAnimaliaDipteraLimoniidae9F0ADF72-CF20-5AA1-80E4-B4473CD6363Chttps://zoobank.org/163BF75E-826C-4E64-AAE4-CFAA23F2E22DHolotype: Korea • ♂; Jeju-do, Seogwipo-si, Hawon-dong, Mt. Hallasan; 33°20.95'N, 126°29.72'E; alt. 1220 m; 13 Jun.–4 Aug. 2021; Y. J. Bae leg.; Malaise trap; GenBank: OM102979; CF21-0148; NIBR.Paratypes: Korea • 1 ♂; Gyeonggi-do, Gapyeong-si, Buk-myeon, Jeokmok-ri, Garim-gyo (Br.); 37°58.60'N, 127°26.55'E; alt. 300 m; 25 Jul.–1 Aug. 2015; Y. J. Bae leg.; Malaise trap; published as D. (E.) tabashii by KUEM • 2 ♂♂, 1 ♀; same data as for preceding; 2–8 Aug. 2015; published as D. (E.) tabashii by KUEM • 1 ♂; same data as for preceding; 23–29 Jul. 2016; published as D. (E.) tabashii by KUEM • 1 ♂; Gangwon-do, Inje-gun, Girin-myeon, Bangdong-ri, Mt. Bangtaesan; 37°54.50'N, 128°24.41'E; alt. 690 m; 30 Jul.–16 Sep. 2019; Y. J. Bae leg.; Malaise trap; KUEM • 1 ♂; Gyeonsangnam-do, Sancheong-gun, Sicheon-myeon, Jungsan-ri, Jungsan-ri Campsite, Mount Jirisan; 35°18.63'N, 127°45.09'E; alt. 700 m; 28 Jul. 2021; J. Kim, C. Lim, D. Lee, W. Lee leg.; sweeping; GenBank: OP081140; CF21-0152; KUEM.Palpus is 3-segmented. Center of male seventh sternite has a deep conical internal sac that has a wide, round entrance. Outer face of gonostylus has two setae arising from a small tubercle. Paramere is elongated and narrow, distally with a darkened tip.Male (holotype). Body length 3.5 mm, wing length 4.5 mm, antenna length 0.7 mm. General body coloration yellow . General body coloration lighter than male.Female terminalia  globulithorax on Mount Bangtaesan and with D. (E.) tabashii on Mount Jirisan.This species is found along intermittent mountain streams in moist mixed forests with grassy vegetation Fig.  and in wAdults are mainly active from July through August.D. (E.) koreana sp. nov. is similar to D. (E.) tabashii, but it can be distinguished by the following characters: palpus 3-segmented (vs 2-segmented); male seventh sternite with weakly darkened, conical internal sac with round entrance ; paramere with darkened tip (vs without). This species is also similar to another species, D. (E.) jejuensis sp. nov. based on the male genital structures, but it can be distinguished by the following characters: male seventh sternite with a deep, conical internal sac ; gonostylus with two setae from tubercle (vs a single seta); paramere without hook at tip (vs with hook).In terms of the shape of the male terminalia, D. (E.) koreana sp. nov. from Mount Bangtaesan differs from other materials of the species based on the shape of the seventh sternite internal sack  and paramere distal lobe (pointed tip). However, additional specimens are needed to determine whether this difference is due to intra- or interspecific variation.The male genitalia of COI sequences contained 190 variable sites, of which 156 were parsimony-informative. The interspecific divergences (p-distances) within subgenus D. (Erostrata) ranged from 11.54% to 16.42%, with a mean distance of 13.17% across the entire dataset (Table D. (E.) jejuensis sp. nov., 0.59% in D. (E.) koreana sp. nov., and 0.15% in D. (E.) tabashii. The maximum intraspecific genetic distance (0.59%) was much smaller than the minimum interspecific one (11.54%). The NJ tree   species. The present study identified two new species using both morphological and molecular data. According to the NJ tree  jejuensis sp. nov., D. (E.) koreana sp. nov., D. (E.) tabashii, and D. (E.) yazuensis can be distinguished from other members of their subgenus based on the shape and mesal face  of their gonostyli. Two hypotheses may be considered: i) this clade can be classified into morphological species groups, or ii) it can be elevated to a new subgenus. Additional materials are needed to more accurately reconstruct phylogenetic relationships within genus Dicranomyia.This is the first study to use DNA barcoding for the delimitation of the ree Fig. , the subD. (E.) tabashii by D. (E.) koreana sp. nov. Based on our observation, unknown cryptic species of crane flies could also be detected and identified using molecular data.Based on our morphological examinations of the materials, we also found that some specimens identified as


Granulomatous polyangiitis (GPA) is a rare autoimmune disease that can involve multiple systems throughout the body, including the ear, nose, upper and lower respiratory tracts. It is classified as an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Telitacicept is a novel recombinant fusion protein targeting B-lymphocyte stimulator (BLyS). Telitacicept can inhibit the development and maturation of abnormal B cells by blocking BLyS, and inhibit the production of antibodies by abnormal plasma cells by blocking APRIL (A proliferation-inducing ligand), which is expected to become a new drug for the treatment of GPA. We report a 64-year-old man diagnosed at our hospital with GPA involving multiple systems including kidneys, lungs, nose and ears. Renal involvement was severe, with a clinical characteristic of rapidly progressive glomerulonephritis and a pathologic manifestation of crescentic nephritis with plasma cell infiltration. The patient was treated with hormones, immunoglobulins and cyclophosphamide (CYC) with the addition of telitacicept and a rapid reduction in hormone dosage. The patient’s renal function improved significantly within a short period of time, and his hearing and lung lesions improved significantly. At the same time, he did not develop serious infections and other related complications. Our report suggests that short-term control of the patient’s conditions is necessary in GPA patients with organ-threatening disease. Telitacicept combined with CYC and glucocorticoids may be an induction therapy with safety and feasibility. However, more clinical trials are needed to validate the efficacy and safety of the therapeutic regimen. Necrotizing vasculitis primarily involves small and medium-sized vessels and can involve life-threatening organs, such as alveolar hemorrhage. Rapidly progressive glomerulonephritis which manifests pathologically as crescentic glomerulonephritis, carries a high risk of progression to end stage renal disease (ESRD) or dialysis-dependent risk . GPA is Compared with normal people, naive B cells increased during AVV activity and GPA remission period , 5, and As we reported below, we observed favorable safety and efficacy of telitacicept in combination with conventional therapies  for the treatment of patients with severe, rapidly progressive renal impairment who with pathological manifestations of crescentic nephritis with plasma cell infiltration.22. He developed hearing loss and no purulent discharge in the external ear canal. No obvious abnormality was found in cardiopulmonary examination. Admission laboratory examination: Blood examination: white blood cells (WBC) 12.13 ×10^9/L (reference range 3.5-9.5*10^9/L), hemoglobin (Hb) 90 g/L (reference range 130-175 g/L); Urine examination: 5-10 red blood cells/HP were detected by microscopy (reference range 0-2/HP); 24-hour urinary protein quantification: 0.5-0.7 g/24h (reference range 0-150 mg/24h); Erythrocyte sedimentation rate (ESR): 29 mm/h (reference range 0-43.5 mm/h) C-reactive protein 118 mg/L (reference range<10 mg/L); Biochemical indicators: Urea 16.3 mmol/L (reference range 3.1-8.0 mmol/L), Creatinine (CRE) 382 μmol/L (reference range 57-97 umol/L), albumin 27 g/L (reference range 40-55 g/L). pANCA (+/-) (reference range -), MPO (++) (reference range -); Immunoglobulin indicators: IgG 19.9 g/L (reference range 7.0-16.0 g/L), IgA 1.35 g/L (reference range 0.7-4.0 g/L), IgM 0.436 g/L (reference range 0.4-2.3 g/L). Imaging examination: Lung CT: 1. Multiple nodules and spots in both lungs, and neoplastic lesions cannot be excluded; 2. Multiple lymph nodes enlargement in the mediastinum  see Figure 13Systemic vasculitis diseases are considered as diseases with multisystem involvement. GPA commonly involves the ear, nose, upper and lower respiratory tracts . In 2022It has been shown that GPA is predominantly characterized by C-ANCA antibody positivity 73.3-76%) and PR-3 ANCA 74-82.2%) antibody positivity, and also by P-ANCA antibody positivity (9.8-13%) and MPO-ANCA antibody positivity (5-8.1%) .The datasets presented in this article are not readily available because this is a case report. Requests to should be directed to Liqi Huang  for the publication of any potentially identifiable images or data included in this article.LH: Investigation, Project administration, Writing – original draft. WL: Investigation, Visualization, Writing – original draft. YLiu: Investigation, Methodology, Writing – original draft. JZ: Resources, Writing – review & editing. YLi: Conceptualization, Resources, Writing – review & editing. ZZ: Conceptualization, Funding acquisition, Writing – review & editing. CT: Conceptualization, Funding acquisition, Writing – review & editing.


Correction: Clinical Epigenetics (2023) 15:105 10.1186/s13148-023-01521-wThe wrong Additional file 4 was originally published with this article . It has The original article has been corrected.Additional file 4. Table S6. Application for NNLS model with 6 CpGs.


Scientific Reports 10.1038/s41598-022-15180-z, published online 07 July 2022Correction to: The original version of this Article contained an error in the Acknowledgements section.“I.I. acknowledges the partial financial support from the SONATA BIS project 2020/38/E/ST5/00176.”now reads:“I.I. was partly funded by the NCN SONATA-BIS Program (UMO-2020/38/E/ST5/00176).”The original Article has been corrected.


Dear Editor,. Some previous studies had suggested an association between higher interleukin(IL)-6 levels and mortality risk in prevalent HD patients. The influence of serum IL-6levels on nutritional status remains, however, to be elucidated. The aim of this studywas to evaluate the association between serum IL-6 levels and nutritional status, andits impact on short-term mortality in prevalent HD patients.Inflammation has been associated with increased mortality in hemodialysis (HD) patients+), and calcium (Ca2+). PTH was measured byelectrochemiluminescence and IL-6 levels were measured by ELISA. Baselinecharacteristics and laboratory data were compared among groups using the Student’st-test for normally distributed continuous variables, Mann-Whitney U-test for skeweddistributed continuous variables, and Chi-square test for categorical variables.One-year all-cause mortality was assessed using standard survival methods. Statisticalanalysis was performed using SPSS (Version 23 for Mac OSX).We conducted a retrospective single-center study of prevalent HD patients between January2020 and January 2021. Patients with acute or chronic inflammatory or infectious diseaseand active malignancy were excluded. According to the mean IL-6 level measured atbaseline, patients were categorized into two cohorts: IL-6 ≤ 17pg/mL and IL-6 >17pg/mL. Baseline clinical and demographic data were collected. Laboratory parameterswere measured before the second HD session of the week, including hemoglobin (Hgb),ferritin, serum iron, transferrin saturation index (TSI), albumin, C-reactive protein(CRP), phosphorus (Pp = 0.045), had higher c-reactive protein(CRP) , lower serum iron, and lower transferrinsaturation index (TSI) .Those patients had also higher serum ferritin levels, although not statisticallysignificant . Regarding nutritional parameters, patients with IL-6 > 17 pg/mL had loweralbumin , lean mass index(LMI) , bodymass index (BMI) , and cholesterol , but without statistical significance. During the study follow-up period, 9(14.8%) patients died. One-year survival was 91.4% in the group with IL-6 ≤ 17 pg/mL and64.3% in those with IL-6 > 17 pg/mL .Using a Cox proportional hazards analysis, we observed that patients with IL-6 > 17pg/mL had an increased risk of all-cause mortality at one year compared to those withIL-6 ≤ 17 pg/mL .The mean age of the included 61 prevalent HD patients was 74.96 ± 12.89 years, 34 (55.7%)were male, and 25 (41%) were diabetic. Mean IL-6 level was 17.3 ± 28.19 pg/mL. Fourteenpatients (23%) had IL-6 > 17pg/mL. There was no gender, comorbidity, HD vintage, ormodality difference among groups. Patients with IL-6 > 17pg/mL were older , and cardiovascularmorbidity to be interrelated in HD patients, each additionally contributing to highermortality in prevalent HD patients.


Correction: Psicol. Refl. Crít. 36, 19 (2023) https://doi.org/10.1186/s41155-023-00262-2Following publication of the original article (Fitzpatrick et al., Maira Almeida Lopes to Maíra Lopes Almeida, Giana Frizzo to Giana Bitencourt FrizzoThe original article (Fitzpatrick et al.,


Long-acting cabotegravir and rilpivirine (CAB/RPV) offers a promising alternative to daily oral antiretroviral therapy (ART); however, system-level and individual challenges in wide scale implementation are anticipated. We describe a community, infusion center-based model (ICBM) for administration of CAB/RPV and associated clinical outcomes.This was a single-center, retrospective cohort study of adults with HIV who were referred for enrollment in the ICBM  from 3/1/22 to 2/28/23 . We investigated demographics, system-level implementation variables, individual factors, and clinical outcomes among CAB/RPV recipients.79 patients were referred for enrollment in the ICBM and 64 patients received at least 1 dose of CAB/RPV. Reasons for CAB/RPV not being administered include insurance barrier, N=2 (13.3%); changed mind, N=3 (20%); unable to reach, N=2 (13.3%), difficulty with collecting labs, N=1 (6.7%), and CAB/RPV started outside of study period, N=4 (26.7%). Of those who received at least one dose of CAB/RPV, 79.9% were male, 48.4% were African American, 42.4% were Medicaid beneficiaries, 67.2% reported mental illness and 39.1% reported alcohol or non-tobacco substance use. Other baseline factors are listed in Table 1. Mean time from referral to first injection was 38.8 days excluding oral lead in time. Mean treatment duration was 176 days (range 20-326 days). 211 maintenance injections were administered during the study period; 16 (7.6%) were outside of the injection window with 10 (62.5%) oral bridges administered. All patients were virally suppressed at end of study period. Overall, 1,122 interventions were completed . Table 2 describes interventions performed.Implementation of CAB/RPV requires a collaborative effort to address system-level and individual challenges. Utilizing existing infrastructure allows for resource optimization to engage vulnerable populations and enhance equitable access. Our program shows successful treatment with CAB/RPV in individuals with adherence barriers (Table 3) at community infusion centers.Carlos Malvestutto, MD MPH, Pfizer: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant


Outpatient parental antibiotic therapy is safe and effective. Our institution is one of two tertiary public hospital pediatric centers in Singapore. We aim to describe characteristics and outcomes of pediatric OPAT (p-OPAT).We retrospectively reviewed p-OPAT patients from May 2013-Dec 2022. Clinically stable inpatients were referred to the pediatric infectious disease team for assessment, caregiver training, and weekly review. Two main delivery models were used; daily bolus/short infusion at an outpatient unit, and home self-administration via elastomeric device. Electronic medical records were analyzed for demographics, clinical and microbiological data, treatment, and outcomes. Institutional review board approval and written informed consent were waived.219 patients completed 249 p-OPAT episodes; 46.6% female, 53.3% male. Median age was 8 years 5 months (range 11 days-29 years). Patient ethnicities were 53.9% Chinese, 12.3% Indian, 20.5% Malay, 13.3% others.53/219 (24.2%) patients were immunocompromised; three had primary immunodeficiency, 50 had acquired immunodeficiency. 89/219 (40.6%) patients had chronic illness.176/249 (70.7%) episodes had only one pathogen identified (Tab 1). Pathogens were gram positive (21.7%), gram negative (38.2%), fungal (5.2%), mycobacterial (5.6%) (Tab 2). 18.9% episodes were culture-negative, 1.6% episodes had no cultures. Infection sites varied considerably; osteoarticular infection +/- bacteremia were most common (Tab 3). Ceftriaxone was the most frequent antimicrobial . Peripherally inserted central catheter was most commonly used .Complete success, partial success, and failure were seen in 174 (69.9%), 43 (17.3%) and 32 (12.8%) episodes respectively. Main adverse events (AE) were vascular site dermatitis, infection, and line dislodgement. 235 (94.4%) episodes achieved infection cure. Mean p-OPAT duration was 16.9 days (range 1-177 days), saving 4208 admission bed days.We describe one of the largest series of children receiving p-OPAT over 10 years and report good outcomes for a broad spectrum of infections. Vascular access issues were common AEs despite preventive measures.All Authors: No reported disclosures


The use of medical 3D printing has expanded dramatically for breast diseases. A writing group composed of the Radiological Society of North America (RSNA) Special Interest Group on 3D Printing (SIG) provides updated appropriateness criteria for breast 3D printing in various clinical scenarios. Evidence-based appropriateness criteria are provided for the following clinical scenarios: benign breast lesions and high-risk breast lesions, breast cancer, breast reconstruction, and breast radiation (treatment planning and radiation delivery).The online version contains supplementary material available at 10.1186/s41205-023-00171-1. Currently, medical 3D printing is performed for a variety of indications. In 2018, the RSNA 3D printing SIG published appropriateness criteria for medical 3D printing for various clinical scenarios [The RSNA Special Interest Group (SIG) on 3D Printing Guidelines Committee has initiated and revised several documents regarding the Clinical scenarios for which 3D Printing is considered an appropriate representation or extension of data contained in a medical imaging examination. This document highlights appropriateness of medical 3D printing for clinical utilization, research, scientific and informational purposes within breast diseases. This work is loosely modeled after the American College of Radiology (ACR) Appropriateness Criteria®  in that 1–3, rarely appropriate. There is a lack of a clear benefit or experience that shows an advantage over usual practice.4–6, maybe appropriate. There may be times when there is an advantage, but the data is lacking, or the benefits have not been fully defined.7–9, usually appropriate. Data and experience show an advantage to 3D printing as a method to represent and/or extend the value of data contained in the medical imaging examination.The SIG Guidelines Chairperson oversees the ratings via a vote among Special Interest Group members at in-person meetings. The results of the ratings follow the following 1–9 format (with 9 being the most appropriate):Clinical scenarios included in this document are stratified by histopathologic diagnosis and treatment. These include benign breast lesions and high-risk breast lesions, malignant breast lesions, breast reconstruction, and breast radiation (treatment planning and radiation delivery).th, 2022 SIG Appropriateness Committee Meeting. Afterwards, 2-week period for comments by SIG members was posted on the SIG’s members-only online forum. All included studies were graded with a strength of evidence assessment according to ACR Appropriateness Criteria Evidence Document.An exhaustive English language PubMed literature search was performed (May 2022) which enabled the querying and retrieval of pertinent clinical documents regarding the appropriateness of 3D printing in each of the scenarios. The supporting evidence was obtained through structured searches, as detailed in each category. For each category, from the pool of total results, the number of publications considered “relevant results” was curated by consensus between physicians with expertise in 3D printing and breast care. Relevant publications that were not retrieved by the structured PubMed search were manually entered. The following categories were excluded because they were considered outside the project scope . All final components of this section were vetted and approved by vote of Special Interest Group members virtually at the July 20Benign breast lesions and high-risk breast lesions: Benign breast diseases are common and include a wide range of entities [entities . The mosentities . Fibroadentities . Benign entities , 4.High-risk breast lesions may confer an increased risk for breast cancer, may be associated with a higher risk for future breast cancer, and may be precursors in breast carcinogenesis. Management of high-risk lesions after core needle biopsy may include close imaging and clinical follow-up or excisional biopsy to evaluate for cancer –8. High-Surgical management of these entities may be needed in cases where cosmesis is altered or when symptom relief is needed. Surgical management may impact developing breast tissue in young women leading to alterations in its proper development . TherefoPubMed Search: ((3D printing) AND (fibrocystic change)) OR ((3D printing) AND (benign breast masses)) OR ((3D printing) AND (mastitis)) OR ((3D printing) AND ) OR ((rapid prototyping) AND (fibrocystic change)) OR ((rapid prototyping) AND (benign breast masses)) OR ((rapid prototyping) AND (mastitis) OR ((rapid prototyping) AND ). ((3D printing) AND ) OR ((3D printing) AND ) OR ((3D printing) AND (lobular neoplasia)) OR ((3D printing) AND ) OR ((3D printing) AND (papillary lesions) OR ((3D printing) AND (mucocele-like lesions)) OR ((rapid prototyping) AND ) OR ((rapid prototyping) AND ) OR ((rapid prototyping) AND (lobular neoplasia) OR ((rapid prototyping) AND ) OR ((rapid prototyping) AND (papillary lesions)) OR ((rapid prototyping) AND (mucocele-like lesions))PubMed Results: No results found.Preliminary rating: 1Malignant breast lesions: Breast cancer is the most common solid malignancy in women in the United States [d States . Approxid States , 12. Bred States . Understd States . They ard States . 3D prind States . This ind States –19. AddiPubMed Search: ((3D printing) AND (breast cancer) OR ((rapid prototyping) AND (breast cancer))PubMed Results: 14 [aSchulz-Wendtland R, Harz M, Meier-Meitinger M, et al. Semi-automated delineation of breast cancer tumors and subsequent materialization using three-dimensional printing (rapid prototyping). J Surg Oncol. 2017;115(3):238–242.bSantiago L, Volk RJ, Checka CM, et al. Acceptability of 3D‐printed breast models and their impact on the decisional conflict of breast cancer patients: A feasibility study. Journal of surgical oncology. 2021;123(5):1206–1214.cBarth RJ, Krishnaswamy V, Paulsen KD, et al. A Patient-Specific 3D-Printed Form Accurately Transfers Supine MRI-Derived Tumor Localization Information to Guide Breast-Conserving Surgery. Annals of Surgical Oncology. 2017;24(10):2950–2956.dRao N, Chen K, Yang Q, Niu J. Proof-of-Concept Study of 3-D-Printed Mold-Guided Breast-Conserving Surgery in Breast Cancer Patients. Clin Breast Cancer. 2018;18(5):e769-e772.eKo BS, Kim N, Lee JW, et al. MRI-based 3D-printed surgical guides for breast cancer patients who received neoadjuvant chemotherapy. Scientific Reports. 2019;9(1):11,991.fFernandez RAS, Lau RWH, Yu PSY, Siu ICH, Chan JWY, Ng CSH. Use of custom made 3-dimensional printed surgical guide for manubrio-sternal resection of solitary breast cancer metastasis: case report. AME Case Rep. 2020;4:12.gWu ZY, Alzuhair A, Kim H, et al. Magnetic resonance imaging based 3-dimensional printed breast surgical guide for breast-conserving surgery in ductal carcinoma in situ: a clinical trial. Sci Rep. 2020;10(1):18,534.hOck J, Lee S, Kim T, et al. Accuracy evaluation of a 3D printing surgical guide for breast-conserving surgery using a realistic breast phantom. Computers in Biology and Medicine. 2021;137:104,784.iWu ZY, Kim HJ, Lee J, et al. Breast-conserving surgery with 3D-printed surgical guide: a single-center, prospective clinical study. Sci Rep. 2021;11(1):2252.jWu ZY, Kim GB, Choi S, Lee S, Kim N, Ko B. Breast-Conserving Surgery after Neoadjuvant Chemotherapy Using a Three-Dimensional-Printed Surgical Guide Based on Supine Magnetic Resonance Imaging: A Case Report. J Breast Cancer. 2021;24(2):235–240.kWu ZY, Lee YJ, Shin Y, et al. Usefulness of 3-Dimensional-Printed Breast Surgical Guides for Undetectable Ductal Carcinoma In Situ on Ultrasonography: A Report of 2 Cases. J Breast Cancer. 2021;24(3):349–355.lWu ZY, Kim GB, Lee S, Choi SH, Kim N, Ko B. Case Report: A 3D-Printed Surgical Guide for Breast-Conserving Surgery After Neoadjuvant Chemotherapy. Front Oncol. 2021;11:633,302.mLee HS, Kim HJ, Chung IY, et al. Usefulness of 3D-surgical guides in breast conserving surgery after neoadjuvant treatment. Sci Rep. 2021;11(1):3376.nSantiago L, Adrada BE, Caudle AS, Clemens MW, Black DM, Arribas EM. The role of three‐dimensional printing in the surgical management of breast cancer. Journal of surgical oncology. 2019;120(6):897–902.ults: 14 , 18–29aSPreliminary Rating: 7Breast reconstruction: Breast reconstruction surgeries include either implant-based or autologous flap reconstructions. In autologous flap reconstructions, 3D printed models have been shown to facilitate the intramuscular dissection of perforator vessels by depicting the course and trajectory of the subfascial vascular tree and allowing the surgeon to view the model from various vantage points [e points , 30, 31.e points . 3D modee points , 34.PubMed Search: ((3D printing) AND (breast reconstruction) OR ((rapid prototyping) AND (breast reconstruction)PubMed Results: 11 [aJablonka EM, Wu RT, Mittermiller PA, Gifford K, Momeni A. 3-DIEPrinting: 3D-printed models to assist the intramuscular dissection in abdominally based microsurgical breast reconstruction. Plastic and Reconstructive Surgery Global Open. 2019;7(4).bChae MP, Rozen WM, McMenamin PG, Findlay MW, Spychal RT, Hunter-Smith DJ. Emerging applications of bedside 3D printing in plastic surgery. Frontiers in surgery. 2015;2:25.cChae MP, Hunter-Smith DJ, Rostek M, Smith JA, Rozen WM. Enhanced preoperative deep inferior epigastric artery perforator flap planning with a 3D-printed perforasome template: technique and case report. Plastic and Reconstructive Surgery Global Open. 2018;6(1).dChae MP, Hunter-Smith DJ, Spychal RT, Rozen WM. 3D volumetric analysis for planning breast reconstructive surgery. Breast Cancer Res Treat. 2014;146(2):457–460.eHummelink S, Verhulst AC, Maal TJ, Ulrich DJ. Applications and limitations of using patient-specific 3D printed molds in autologous breast reconstruction. European journal of plastic surgery. 2018;41(5):571–576.fDeFazio MV, Arribas EM, Ahmad FI, et al. Application of Three-Dimensional Printed Vascular Modeling as a Perioperative Guide to Perforator Mapping and Pedicle Dissection during Abdominal Flap Harvest for Breast Reconstruction. J Reconstr Microsurg. 2020;36(5):325–338.gMehta S, Byrne N, Karunanithy N, Farhadi J. 3D printing provides unrivalled bespoke teaching tools for autologous free flap breast reconstruction. Journal of Plastic, Reconstructive & Aesthetic Surgery. 2016;69(4):578–580.hChae MP, Hunter-Smith DJ, Chung RD, Smith JA, Rozen WM. 3D-printed, patient-specific DIEP flap templates for preoperative planning in breast reconstruction: a prospective case series. Gland Surg. 2021;10(7):2192–2199.iChen K, Feng CJ, Ma H, et al. Preoperative breast volume evaluation of one-stage immediate breast reconstruction using three-dimensional surface imaging and a printed mold. J Chin Med Assoc. 2019;82(9):732–739.jOgunleye AA, Deptula PL, Inchauste SM, et al. The utility of three-dimensional models in complex microsurgical reconstruction. Arch Plast Surg. 2020;47(5):428–434.kTomita K, Yano K, Taminato M, Nomori M, Hosokawa K. DIEP Flap Breast Reconstruction in Patients with Breast Ptosis: 2-Stage Reconstruction Using 3-Dimensional Surface Imaging and a Printed Mold. Plast Reconstr Surg Glob Open. 2017;5(10):e1511.ults: 11 –40aJabloPreliminary rating: 8Breast Radiation (Treatment planning and Radiation delivery): 3D printing can be used to design customized patient specific boluses and shields that allow homogeneous distribution of radiation dose to the area of interest while sparing adjacent normal tissue [l tissue . 3D prinl tissue PubMed Search: ((3D printing) AND (breast radiation) OR ((rapid prototyping) AND (breast radiation)PubMed Results: 8 [aPoulin E, Gardi L, Fenster A, Pouliot J, Beaulieu L. Towards real-time 3D ultrasound planning and personalized 3D printing for breast HDR brachytherapy treatment. Radiother Oncol. 2015;114(3):335–338.bAristei C, Lancellotta V, Piergentini M, et al. Individualized 3D-printed templates for high-dose-rate interstitial multicathether brachytherapy in patients with breast cancer. Brachytherapy. 2019;18(1):57–62.cYang K, Park W, Ju SG, et al. Heart-sparing radiotherapy with three-dimensional printing technology after mastectomy for patients with left breast cancer. Breast J. 2019;25(4):682–686.dHe C, Zhang S, Shi L. Three-Dimensionally-Precise Breast Conformal Device for IMRT in Breast Cancer Patients Treated With Breast-Conserving Surgery-A Pilot Randomized Controlled Trial. Technol Cancer Res Treat. 2020;19:1,533,033,820,971,563.eRobar JL, Moran K, Allan J, et al. Intrapatient study comparing 3D printed bolus versus standard vinyl gel sheet bolus for postmastectomy chest wall radiation therapy. Pract Radiat Oncol. 2018;8(4):221–229.fPark SY, Choi CH, Park JM, Chun M, Han JH, Kim JI. A Patient-Specific Polylactic Acid Bolus Made by a 3D Printer for Breast Cancer Radiation Therapy. PLoS One. 2016;11(12):e0168063.gPoulin E, Gardi L, Fenster A, Pouliot J, Beaulieu L. A novel approach for real-time, personalized breast HDR brachytherapy treatment using 3D printing technology. Brachytherapy. 2014;13:S18.hPark K, Park S, Jeon MJ, et al. Clinical application of 3D-printed-step-bolus in post-total-mastectomy electron conformal therapy. Oncotarget. 2017;8(15):25,660–25,668.sults: 8 , 42–48aPPreliminary rating: 6Table Limitations of this work include its lack of objective data collection and inferential statistics. Although such an analysis would be desirable, it is not practical with most published breast related applications due to the small number of publications and patients. PubMed search terms, were based on prior search terminology from previously published guidelines . The RSNThis document provides updated appropriateness criteria for 3D printing in breast conditions. Adoption of common clinical standards regarding appropriate use, information and material management, and quality control are needed to ensure the greatest possible clinical benefit from 3D printing. With accruing evidence for in 3D printing, this consensus guideline document, created by the members of the RSNA 3D printing Special Interest Group, will provide a reference for clinical standards of 3D printing. The document will be periodically refined, based on expanding clinical applications and growing medical literature.Additional file 1.