Implanted Tissue-Engineered Vascular Graft Cell Isolation with Single-Cell RNA Sequencing Analysis.

Tissue-engineered vascular grafts (TEVGs) require adequate extracellular matrix (ECM) to withstand arterial pressure. Tissue transglutaminase (TG2) and lysyl oxidase (LOX) are enzymes that cross-link ECM proteins and play a pivotal role in the development of vascular stiffness associated with aging. The purpose of this study is to investigate the expression of ECM cross-linking enzymes and mechanisms of scaffold degeneration leading to vascular stiffness in TEVG remodeling. Fast- and slow-degrading electrospun TEVGs were fabricated using polydioxanone (PDO) and poly(L-lactide-co-caprolactone) (PLCL) copolymer, with a PDO/PLCL ratio of 9:1 for fast-degrading and 1:1 for slow-degrading graft. These grafts were implanted in rats (n = 5/group) as abdominal aortic interposition conduits. The grafts were harvested at 1 month to evaluate patency, mechanical properties, vascular neotissue formation, and the expression of ECM cross-linking enzymes. All TEVGs were patent without any aneurysmal formation at 1 month. ECM area, TG2-positive area, and LOX-positive area were significantly greater in fast-degrading TEVGs compared to slow-degrading TEVGs, with significantly less remaining scaffold. The mechanical properties of fast-degrading TEVGs were similar to that of native aorta, as demonstrated by strain-stress curve. In conclusion, at 1 month, fast-degrading TEVGs had rapid and well-organized ECM with greater TG2 and LOX expression and native-like mechanical properties, compared to slow-degrading TEVGs. Impact statement Around 1.4 million patients in the United States require arterial prostheses each year due to cardiovascular diseases. Current synthetic vascular grafts suffer from increased risk of infection, thrombosis, a lack of endothelialization, and compliance mismatch to the native vasculature. Tissue-engineered vascular graft (TEVGs) presented in this study exhibited tunable biodegradation profiles by controlling the polymer ratio of polydioxanone/poly(L-lactide-co-caprolactone). One month after implantation, the fast-degrading TEVGs exhibited mechanical properties similar to that of native aorta, formation of endothelium, and well-organized extracellular matrix (ECM) with increased expression of tissue transglutaminase and lysyl oxidases, which are critical to the ECM remodeling process.

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and severe cardiopulmonary disease with a challenging prognosis, and its underlying pathogenesis remains elusive. A comprehensive understanding of IPAH is crucial to unveil potential diagnostic markers and therapeutic targets. In this study, we investigated cellular heterogeneity and molecular pathology in IPAH using single-cell RNA sequencing (scRNA-seq) analysis. Our scRNA-seq results revealed significant alterations in three crucial signaling pathways in IPAH: the hypoxia pathway, TGF β pathway, and ROS pathway, primarily attributed to changes in gene expression within arterial endothelial cells. Moreover, through bulk RNA sequencing analysis, we identified differentially expressed genes (DEGs) enriched in GO and KEGG pathways, implicated in regulating cell adhesion and oxidative phosphorylation in IPAH lungs. Similarly, DEGs-enriched pathways in IPAH arterial endothelial cells were also identified. By integrating DEGs from three IPAH datasets and applying protein-protein interaction (PPI) analysis, we identified 12 candidate biomarkers. Subsequent validation in two additional PAH datasets led us to highlight five potential biomarkers (CTNNB1, MAPK3, ITGB1, HSP90AA1, and DDX5) with promising diagnostic significance for IPAH. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) confirmed significant differences in the expression of these five genes in pulmonary arterial endothelial cells from PAH mice. In conclusion, our findings shed light on the pivotal role of arterial endothelial cells in the development of IPAH. Furthermore, the integration of single-cell and bulk RNA sequencing datasets allowed us to pinpoint novel candidate biomarkers for the diagnosis of IPAH. This work opens up new avenues for research and potential therapeutic interventions in IPAH management.

Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells

Background:The production of tissue-engineered vascular graft (TEVG) usually involves a prolonged bioreactor cultivation period of up to several weeks to achieve maturation of extracellular matrix and sufficient mechanical strength. Therefore, we aimed to substantially shorten this conditioning time by combining a TEVG textile scaffold with a recently developed copolymer reinforced fibrin gel as a cell carrier. We further implemented our grafts with magnetic resonance imaging (MRI) contrast agents to allow the in-vitro monitoring of the TEVG’s remodeling process.Methods:Biodegradable polylactic-co-glycolic acid (PLGA) was electrospun onto a non-degradable polyvinylidene fluoride scaffold and molded along with copolymer-reinforced fibrin hydrogel and human arterial cells. Mechanical tests on the TEVGs were performed both instantly after molding and 4 days of bioreactor conditioning. The non-invasive in vitro monitoring of the PLGA degradation and the novel imaging of fluorinated thermoplastic polyurethane (19F-TPU) were performed using 7T MRI.Results:After 4 days of close loop bioreactor conditioning, 617 ± 85 mmHg of burst pressure was achieved, and advanced maturation of extracellular matrix (ECM) was observed by immunohistology, especially in regards to collagen and smooth muscle actin. The suture retention strength (2.24 ± 0.3 N) and axial tensile strength (2.45 ± 0.58 MPa) of the TEVGs achieved higher values than the native arteries used as control. The contrast agents labeling of the TEVGs allowed the monitorability of the PLGA degradation and enabled the visibility of the non-degradable textile component.Conclusion:Here, we present a concept for a novel textile-reinforced TEVG, which is successfully produced in 4 days of bioreactor conditioning, characterized by increased ECM maturation and sufficient mechanical strength. Additionally, the combination of our approach with non-invasive imaging provides further insights into TEVG’s clinical application.

In-vivo assessment of a tissue engineered vascular graft computationally optimized for target vessel compliance

Bioresorbable silk grafts for small diameter vascular tissue engineering applications: In vitro and in vivo functional analysis

Background: Lactylation is closely involved in cancer progression, but its role in hepatocellular carcinoma (HCC) is unclear. The present work set out to develop a lactylation-related gene (LRG) signature for HCC. Methods: The lactylation score of tumor and normal groups was calculated using the gene set variation analysis (GSVA) package. The single-cell RNA sequencing (scRNA-seq) analysis of HCC was performed in the "Seurat" package. Prognostic LRGs were selected by performing univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses to develop and validate a Riskscore model. Functional enrichment analysis was conducted by gene set enrichment analysis (GSEA) using the "clusterProfiler" package. Genomic characteristics between different risk groups were compared, and tumor mutational burden (TMB) was calculated by the "Maftools" package. Immune cell infiltration was assessed by algorithms of cell-type identification by estimating relative subsets of RNA transcript (CIBERSORT), microenvironment cell populations-counter (MCP-counter), estimating the proportions of immune and cancer cells (EPIC), tumor immune estimation resource (TIMER), and single-sample gene set enrichment analysis (ssGSEA). Immunotherapy response was predicted by the tumor immune dysfunction and exclusion (TIDE) algorithm. Drug sensitivity was analyzed using the "pRRophetic" package. A nomogram was established using the "rms" package. The expressions of the prognostic LRGs in HCC cells were verified by in vitro test, and cell counting kit-8 (CCK-8), wound healing, and transwell assays were carried out to measure the viability, migration, and invasion of HCC cells. Results: The lactylation score, which was higher in the tumor group than in the normal group, has been confirmed as an independent factor for the prognostic evaluation in HCC. Six prognostic LRGs, including two protective genes (FTCD and APCS) and four risk genes (LGALS3, C1orf43, TALDO1, and CCT5), were identified to develop a Riskscore model with a strong prognostic prediction performance in HCC. The scRNA-seq analysis revealed that LGALS3 was largely expressed in myeloid cells, while APCS, FTCD, TALDO1, CCT5, and C1orf43 were mainly expressed in hepatocytes. The high-risk group was primarily enriched in the pathways involved in tumor occurrence and development, with higher T cell infiltration. Moreover, the high-risk group was found to be less responsive to immunotherapy but was more sensitive to chemotherapeutic drugs. By integrating Riskscore and clinical features, a nomogram with a high predictive accuracy was developed. Additionally, C1orf43, CCT5, TALDO1, and LGALS3 were highly expressed in HCC cells. Silencing CCT5 inhibited the viability, migration, and invasion of HCC cells. Conclusion: The present work developed a novel LRG gene signature that could be considered a promising therapeutic target and biomarker for HCC.

BackgroundGastric cancer (GC) is a leading cause of cancer-related deaths worldwide, with early diagnosis remaining a significant challenge. Available serum biomarkers lack specificity, making it difficult to accurately identify early non-metastatic GC cases. Reliable diagnostic biomarkers that can detect early GC are critical to improve prognosis.MethodsWe employed serum proteomics combined with bioinformatics to identify genes differentially expressed in the serum of non-metastatic GC patients. Single-cell RNA sequencing (ScRNA-seq) and immune infiltration analysis were performed to evaluate the relationship between gene expression and immune cell function. Then we evaluated 107 machine learning models for biomarker-based early GC diagnosis and develops a nomogram validated for accuracy and clinical utility, subsequently comparing the performance of potential biomarkers with traditional tumor markers in diagnosing early gastric cancer. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and immunohistochemical staining using the Human Protein Atlas (HPA) database were used to validate the differential expression of candidate genes in GC tissues and adjacent non-cancerous tissues.ResultsThe proteomic analysis identified several genes upregulated in the serum of GC patients compared to healthy controls. Single-cell RNA sequencing analysis further revealed that these upregulated genes were associated with altered immune cell infiltration in the tumor microenvironment. The glmBoost + XGBoost model incorporating B2M, CFL1, CTSD, and HSP90AB1 demonstrated strong diagnostic performance (mean AUC = 0.792), with 101 algorithm combinations achieving an average AUC > 0.7. A nomogram integrating gene expression and clinical data was developed, validated through calibration and decision curve analyses, highlighting its potential for early GC diagnosis. Additionally, four genes—TAGLN2, HSP90AB1, SH3BGRL3, and CFL1—were found to be highly expressed in non-metastatic GC tissues and were significantly correlated with immune infiltration, including CD8 + T cells, monocytes, and myeloid-derived suppressor cells. These findings were validated by qRT-PCR and immunohistochemical analyses, confirming their elevated expression in GC tissues.ConclusionsTAGLN2, HSP90AB1, SH3BGRL3 and CFL1 are potential diagnostic biomarkers for early-stage GC, with strong associations with immune cell infiltration. Machine learning model shows excellent diagnostic performance. These results provide a foundation for future studies to improve early diagnosis and individualized treatment strategies for GC.

Nondegradable synthetic polymer vascular grafts used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. Tissue engineering of vascular grafts with autologous stem cells and biodegradable polymeric materials could solve these problems. The present study is aimed to develop a tissue-engineered vascular graft (TEVG) with functional endothelium using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. TEVGs (internal diameter: 10 mm, length: 40 mm) were fabricated by seeding vascular cells differentiated from BMCs onto PGA/PLCL scaffolds and implanted into the abdominal aorta of bone marrow donor dogs (n = 7). Eight weeks after implantation of the TEVGs, the vascular grafts remained patent. Histological and immunohistochemical analyses of the vascular grafts retrieved at 8 weeks revealed the regeneration of endothelium and smooth muscle and the presence of collagen. Western blot analysis showed that endothelial nitric oxide synthase (eNOS) was expressed in TEVGs comparable to native abdominal aortas. This study demonstrates that vascular grafts with significant eNOS activity can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.

Introduction: Circulating tumor cells (CTCs) are liquid biomarkers with high potential for application in precision medicine cancer care. However, no robust methods have been developed to culture CTCs from early-stage cancer patients owing to their rarity and instability. Here, we describe a successful protocol to establish CTC culturing and CTC-derived xenograft (CDX) mouse models from early-stage non-small cell lung cancer (NSCLC) patients. Further, comparative multi-omics characterization of these CDX tumors revealed novel diagnostic/therapeutic target(s). Methods: Surgically resected primary tumor fragments from patients with early stage NSCLCs were subcutaneously (s.c.) implanted to immunodeficient NOD SCID gamma (NSG) mice to generate patient-derived xenograft (PDX) models. Once palpable tumor reached a diameter of ~1.5 cm, animals were euthanized and blood was collected, CTCs were enriched with OncoQuick® and cultured in vitro. These patient-derived CTCs were further stained with anti-human antibodies against CK7, Napsin, TTF-1, CK5/6, PDL1 and CD45. Once human CTCs were confirmed, we then injected expanded CTCs s.c. to naïve NSG mice to establish CDX models. Single cell suspension from CDX tumors was cultured and fibroblasts were depleted using specific beads (Miltenyi biotec) to establish stable CTC lines. Further, advanced proteomics, single cell RNA sequencing (transcriptomics) and whole genome sequencing of patient-matched peritumoral and primary tumor tissues along with PDX and CDX tumors were carried out. Results: Human NSCLC primary tumor fragments implanted in NSG mice were tumorigenic at the s.c. implantation site, metastatic to distant organs, and passaged successfully to naïve NSG generations to maintain the PDX colonies. CTCs enriched from the PDX models were successfully cultured in vitro and positive for human NSCLC-specific markers but negative for human leukocyte marker CD45. Cultured PDX-derived CTCs that were xenografted into naïve NSG mice by s.c. demonstrated tumorigenicity and metastatic potential in CDX models. Stable CTC lines established using CDX tumors sustained >30 passages. Global proteomic profiling of tumor tissues revealed 19 new proteins that were upregulated and 9 proteins that are down regulated. Single cell RNA sequence analysis depicts top rank genes that are overexpressed in tumor cluster. Whole genome sequencing of these samples identified somatic variants that needs further validation. Conclusion and future perspectives: This approach establishes CDX models from early-stage NSCLC patients at a high rate for personalized precision medicine. Models provide a unique opportunity to characterize rare patient-derived CTCs that helps in understanding CTC tumor biology and developing effective anti-cancer therapeutic strategies. Multi-omics characterization of these tissues identified a set of novel genes/proteins that needs to be validated in large patient population. Further, studying tumorigenic potential of established CTC lines after knockdown and overexpression of these genes will provide more information about translating these targets into therapeutics. Citation Format: Kanve N. Suvilesh, Yariswamy Manjunath, Vijay Radhakrishnan, Diego M. Avella, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Guangfu Li, Jussuf T. Kaifi. Multi-omics characterization of tumor tissues from CTC derived xenograft (CDX) mouse models of early-stage NSCLC patients reveals novel diagnostic/therapeutic target(s) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-271.

Simple SummaryImmunotherapy has a unique potential for improving various types of cancer treatment. However, related studies in endometrial carcinoma are lacking. Further studies on the tumor-infiltrating lymphocyte populations are vital. We firstly mapped the immune atlas of lymphocytes in type I endometrial carcinoma via single-cell RNA sequencing, mass cytometry and flow cytometry analysis. Three transcriptionally distinct NK cell subsets with different antitumor functions were identified. Some CD103+ CD8+ T cells were also defined as tissue-resident memory T cells in endometrial carcinoma. Both our retrospective study and analysis of a public repository suggest the correlation between antitumor function of CD103+ CD8+ T cells and EC prognosis. Certain key molecular mechanisms potentially determining the phenotypes of NK cells and CD103+ CD8+ T cells were highlighted and might serve as novel therapeutic targets.Endometrial carcinoma (EC) is a gynecological malignancy with a high incidence; however, thorough studies on tumor-infiltrating lymphocyte (TIL) populations in EC are lacking. We aimed to map the immune atlas of TILs in type I EC via single-cell RNA sequencing (scRNA-seq), mass cytometry and flow cytometry analysis. We found that natural killer (NK) cells and CD8+ T lymphocytes were the major components of TILs in EC patients. We first identified three transcriptionally distinct NK cell subsets, which are likely to possess diverse anti-tumor functions. Additionally, CD103+ cells substantially contributed to the CD8+ T cell population. The signature gene expression of CD103+ CD8+ T cells indicated the tissue residency, immunological memory, and exhaustion properties of this cell subset, which were defined as tissue-resident memory T cells (TRM cells). Moreover, based on scRNA-seq and mass cytometry analysis, we first identified the intrinsic heterogeneity of CD103+ CD8+ T cells that were thought to have a distinct cytotoxicity, cell adhesion and exhaustion status functions. Collectively, distinct subsets of NK cells were found and might shed light on future investigations. CD103+ CD8+ T cell population may be an important immunotherapeutic target in EC and targeting this cell population with combined immunosuppressive therapy might improve the efficacy of immunotherapy for EC.

RATIONALE: Chronic lung allograft dysfunction (CLAD) is an unfortunately common complication of lung transplantation, and is associated with poor long-term prognoses. The onset of anti-donor adaptive immune responses, both cellular and humoral, is highly implicated in the pathogenesis of CLAD. Early innate immune responses have been shown to regulate adaptive immunity, with prior reports implicating roles for natural killer (NK) and natural killer T cells in early inflammatory events that predate CLAD. Here we investigate interferon gamma (IFNγ) secreted by NK cells and NK T cells in the context of lung transplant rejection. METHODS: Unilateral left lung transplantation was performed in mice using B6D2F1/J as a donor and either C57BL/6J (allograft) or B6D2F1/J (isograft) as a recipient. Mice were harvested 7 and 14 days after surgery and processed for flow cytometry, and/or RNA isolation. Lung allograft digests enriched for T cells were subjected to single cell RNA sequencing analyses. RNA isolated from isografts and RAS lung allografts from days 14, 28 and 40 were subjected to RNA sequencing analyses. Single cell suspensions generated from Enzymatically digested lungs and spleens were used to generate single cell suspensions from isograft control mice or allografts, then were enriched for NKp46+ NK and NK T cells. Enriched naïve cells were stimulated with IL-15, IL-18 or both for 24-48h followed by FACS and ELISPOT analyses for intracellular IFNγ production. RESULTS: Single cell RNA sequencing analyses identified Ifng as one of the top upregulated genes in murine RAS allografts. RNAseq revealed temporal over-expression of Ifng mRNA in the allografts. NKp46-enriched NK and NK T cells from naïve lungs stimulated with IL-15, IL-18 or both demonstrated significantly higher IFNg production by FACS and ELISPOT analyses, with the presence of both cytokines demonstrating a synergistic effect. NKp46-enriched cells from murine lung allografts as early as day 7 produced significant intracellular IFNγ. NKp46-enriched splenocytes from murine allografts at day 14, that were primed with IL-15 and stimulated with anti-NK1.1 antibodies demonstrated significant intracellular IFNγ production.-- CONCLUSIONS: Innate immune-derived IFNγ is upregulated in specific subsets of NK cells and NK T cells in both naïve cells in the presence of exogenous stimuli as well as unstimulated NK cells derived from allografts. These findings implicate the role of IFNγ production following lung transplantation and emphasizes the need for future studies to investigate the regulation of immune responses in CLAD.

Objective: To analyze the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing. Methods: This study was an experimental study. The CD34+ cell lineage tracing mouse was produced, and the visualization of CD34+ cells under the fluorescent condition was realized. Six male CD34+ cell lineage tracing mice aged 7-8 weeks (designated as diabetic group) were intraperitoneally injected with streptozotocin to establish a diabetic model, and full-thickness skin defect wounds were prepared on their backs when they reached 13 weeks old. Another 6 male CD34+ cell lineage tracing mice aged 13 weeks (designated as control group) were also subjected to full-thickness skin defect wounds on their backs. On post-injury day (PID) 4, wound tissue was collected from 3 mice in control group and 2 mice in diabetic group, and digested to prepare single-cell suspensions. CD34+ cells were screened using fluorescence-activated cell sorting, followed by single-cell RNA sequencing. The Seurat 4.0.2 program in the R programming language was utilized for dimensionality reduction, visualization, and cell clustering analysis of CD34+ cell types, and to screen and annotate the marker genes for each CD34+ cell subpopulation. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was performed to analyze the differentially expressed genes (DEGs) of CD34+ fibroblasts (Fbs), smooth muscle cells (SMCs), keratinocytes (KCs), and chondrocyte-like cells (CLCs) in the wound tissue of two groups of mice for exploring cellular functions. Results: On PID 4, CD34+ cells in the wound tissue of both groups of mice were consisted of 7 cell types, specifically endothelial cells, Fbs, KCs, macrophages, T cells, SMCs, and CLCs. Among these, Fbs were further classified into 5 subpopulations. Compared with those in control group, the proportions of CD34+ endothelial cells, Fbs subpopulation 1, Fbs subpopulation 4, KCs, and CLCs in the wound tissue of mice were increased in diabetic group, while the proportions of CD34+ Fbs subpopulation 2, Fbs subpopulation 3, and SMCs were decreased. The marker genes for annotating CD34+ CLCs, endothelial cells, Fbs subpopulation 1, Fbs subpopulation 2, Fbs subpopulation 3, Fbs subpopulation 4, Fbs subpopulation 5, KCs, macrophages, SMCs, and T cells were respectively metastasis-associated lung adenocarcinoma transcript 1, fatty acid binding protein 4, Gremlin 1, complement component 4B, H19 imprinted maternally expressed transcript, Dickkopf Wnt signaling pathway inhibitor 2, fibromodulin, keratin 5, CD74 molecule, regulator of G protein signaling 5, and inducible T-cell co-stimulator molecule. KEGG and GO enrichment analysis revealed that, compared with those in control group, DEGs with significant differential expression (SDE) in CD34+ Fbs from the wound tissue of mice in diabetic group on PID 4 were significantly enriched in terms related to inflammatory response, extracellular matrix (ECM) organization, regulation of cell proliferation, and aging (with Pvalues all <0.05), DEGs with SDE in CD34+ SMCs were significantly enriched in terms related to cell migration, apoptotic process, positive regulation of transcription, and phagosome (with P values all <0.05), DEGs with SDE in CD34+ KCs were significantly enriched in terms related to mitochondrial function, transcription, and neurodegenerative diseases (with P values all <0.05), and DEGs with SDE in CD34+ CLCs were significantly enriched in terms related to rhythm regulation, ECM, and viral infection (with P values all <0.05). Conclusions: CD34+ cells display high heterogeneity in the healing process of full-thickness skin defect wounds in both normal mice and diabetic mice. The significantly enriched functions of DEGs with SDE in CD34+ cell subpopulations in the wound tissue of the two mouse groups are closely related to the wound healing process.

An off-the-shelf, small diameter tissue engineered vascular graft (TEVG) would be transformative to surgeons in multiple subspecialties. Herein, the results of a small diameter (ID ≈ 1 mm) vascular graft constructed from resorbable, amino acid-based poly(ester urea) (PEU) are reported. Electrospun PEU grafts of two different wall thicknesses (type A: 250 μm; type B: 350 μm) are implanted as abdominal infra-renal aortic grafts in a severe combined immune deficient/beige mouse model and evaluated for vessel remodeling over one year. Significantly, the small diameter TEVG does not rupture or lead to acute thrombogenic events during the intervals tested. The pilot TEVG in vivo shows long-term patency and extensive tissue remodeling with type A grafts. Extensive tissue remodeling in type A grafts leads to the development of well-circumscribed neovessels with an endothelial inner lining, a neointima containing smooth muscle cells. However, due to slow degradation of the PEU scaffold materials in vivo, the grafts remain after one year. The type B grafts, which have 350 μm thick walls, experience occlusion over the one year interval due to intimal hyperplasia. This study affords significant findings that will guide the design of future generations of small diameter vascular grafts.

First long-term preclinical evaluation of woven vascular grafts made from human cell-assembled extracellular matrix.