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Non-alcoholic steatohepatitis (NASH) with fibrosis eventually leads to cirrhosis and hepatocellular carcinoma. Thus, the development of therapies other than dietary restriction and exercise, particularly those that suppress steatosis and fibrosis of the liver and have a long-term beneficial effect, is necessary. We aimed to evaluate the therapeutic effects of the HMGB1 peptide synthesized from box A using the melanocortin-4 receptor-deficient (Mc4r-KO) NASH model mouse.We performed short- and long-term administration of this peptide and evaluated the effects on steatosis, fibrosis, and carcinogenesis using Mc4r-KO mice. We also analyzed the direct effect of this peptide on macrophages and hepatic stellate cells in vitro and performed lipidomics and metabolomics techniques to evaluate the effect.Although this peptide did not show direct effects on macrophages and hepatic stellate cells in vitro, in the short-term administration model, we could confirm the reduction of liver damage, steatosis, and fibrosis progression. The results of lipidomics and metabolomics suggested that the peptide might ameliorate NASH by promoting lipolysis via the activation of fatty acid β-oxidation and improving insulin resistance. In the long-term administration model, this peptide prevented progression to cirrhosis but retained the steatosis state, that is, the peptide prevents the progression to "burnt-out NASH." This peptide inhibited carcinogenesis by about one-third.This HMGB1 peptide can reduce liver damage, improve fibrosis and steatosis, and inhibit carcinogenesis, suggesting that the peptide would be a new treatment candidate for NASH and can contribute to the long-term prognosis for patients with NASH.

PDF file - 103K, Measurement of the ZZ-scIL-12 on Fc-HVJ-E.

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Purpose: Cancer immunotherapy has encountered many difficulties in the face of the expectation to eradicate cancer, and new breakthroughs are required. We have previously shown that UV-inactivated Sendai virus particles (hemagglutinating virus of Japan envelope; HVJ-E) induce immunity against multiple tumor types. In this study, a novel pseudovirion that stimulates more robust antitumor immunity was designed for cancer treatment.Experimental Design: First, we found that culturing murine splenocytes with HVJ-E in combination with interleukin (IL)-12 resulted in a remarkable increase in IFN-γ production compared with that observed in splenocytes cultured with IL-12 alone. The synergistic effects of HVJ-E and IL-12 on IFN-γ production were caused by viral F proteins independently of HVJ-E fusion activity and not by hemagglutination from hemagglutinin-neuraminidase (HN) proteins. We next constructed HN-depleted HVJ-E expressing the Fc region of immunoglobulin G (IgG) on the envelope and single-chain IL-12 containing the ZZ domain of protein A to produce an IL-12–conjugated HVJ-E particle without hemagglutinating activity.Results: IL-12–conjugated HVJ-E dramatically enhanced the amount of IFN-γ produced by immune cells. Intratumoral injection of IL-12–conjugated HVJ-E eradicated murine melanomas more effectively than injection of wild-type HVJ-E through increased production of melanoma-specific CTLs. IL-12–conjugated HVJ-E preferentially accumulated in the lungs after systemic administration. When small metastatic melanoma foci were formed in the lungs, systemic administration of IL-12–conjugated HVJ-E significantly reduced the number of metastatic foci by inducing local production of IFN-γ in the lungs and generating large numbers of melanoma-specific CTLs.Conclusion: IL-12–conjugated HVJ-E is a promising tool for the treatment of cancers, including lung metastasis. Clin Cancer Res; 19(3); 668–79. ©2012 AACR.

PDF file - 137K, FACS analysis of IFN-γ-positive splenocytes.

PDF file - 137K, Measurement of scIL-12-HVJ-E-mediated CTL activation in the mice bearing lung metastases following treatment with the IFN-γ-neutralizing antibody.

PDF file - 67K, Serum IFN-γ levels induced upon the systemic administration of scIL-12-HVJ-E and the scIL-12 and Fc-HVJ-E mixture.

PDF file - 69K, Inhibition of the F protein by the anti-F neutralizing antibody, and IFN-γ induction upon treatment with scIL-12-HVJ-E.

PDF file - 58K, Sucrose gradient sedimentation of ZZ-scIL-12 and wt-HVJ-E.

Epidermolysis bullosa (EB) is a group of inherited skin and mucosal fragility disorders resulting from mutations in genes encoding basement membrane zone (BMZ) components or proteins that maintain the integrity of BMZ and adjacent keratinocytes. More than 30 years have passed since the first causative gene for EB was identified, and over 40 genes are now known to be responsible for the protean collection of mechanobullous diseases included under the umbrella term of EB. Through the elucidation of disease mechanisms using human skin samples, animal models, and cultured cells, we have now reached the stage of developing more effective therapeutics for EB. This review will initially focus on what is known about blister wound healing in EB, since recent and emerging basic science data are very relevant to clinical translation and therapeutic strategies for patients. We then place these studies in the context of the latest information on gene therapy, read-through therapy, and cell therapy that provide optimism for improved clinical management of people living with EB.

The liver has a high regenerative ability and can induce spontaneous regression of fibrosis when early liver damage occurs; however, these abilities are lost when chronic liver damage results in decompensated cirrhosis. Cell therapies, such as mesenchymal stem cell (MSC) and macrophage therapies, have attracted attention as potential strategies for mitigating liver fibrosis. Here, we evaluated the therapeutic effects of HMGB1 peptide synthesized from box A of high mobility group box 1 protein. Liver damage and fibrosis were evaluated using a carbon tetrachloride (CCl4)-induced cirrhosis mouse model. The effects of HMGB1 peptide against immune cells were evaluated by single-cell RNA-seq using liver tissues, and those against monocytes/macrophages were further evaluated by in vitro analyses. Administration of HMGB1 peptide did not elicit a rapid response within 36 h, but attenuated liver damage after 1 week and suppressed fibrosis after 2 weeks. Fibrosis regression developed over time, despite continuous liver damage, suggesting that administration of this peptide could induce fibrolysis. In vitro analyses could not confirm a direct effect of HMGB1 peptide against monocyte/macrophages. However, macrophages were the most affected immune cells in the liver, and the number of scar-associated macrophages (Trem2+Cd9+ cells) with anti-inflammatory markers increased in the liver following HMGB1 treatment, suggesting that indirect effects of monocytes/macrophages were important for therapeutic efficacy. Overall, we established a new concept for cell-free therapy using HMGB1 peptide for cirrhosis through the induction of anti-inflammatory macrophages.

Background & Aims Proper resolution of inflammation is essential to maintaining homeostasis, which is important as a dysregulated inflammatory response has adverse consequences, even being regarded as a hallmark of cancer. However, our picture of dynamic changes during inflammation remains far from comprehensive. Methods Here we used single-cell transcriptomics to elucidate changes in distinct cell types and their interactions in a mouse model of chemically induced colitis. Results Our analysis highlights the stromal cell population of the colon functions as a hub with dynamically changing roles over time. Importantly, we found that Serpina3n, a serine protease inhibitor, is specifically expressed in stromal cell clusters as inflammation resolves, interacting with a potential target, elastase. Indeed, genetic ablation of the Serpina3n gene delays resolution of induced inflammation. Furthermore, systemic Serpina3n administration promoted the resolution of inflammation, ameliorating colitis symptoms. Conclusions This study provides a comprehensive, single-cell understanding of cell-cell interactions during colorectal inflammation and reveals a potential therapeutic target that leverages inflammation resolution., Graphical abstract

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1−Ibsp+Sparcl1+ and Plap-1−Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

Background Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use. Objective In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation. Methods For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression. Results After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their “stemness”. When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored. Conclusions Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations-two distinct mutations on each chromosome-without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin− cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin− HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin− HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Mesenchymal stem cells (MSCs), which can differentiate into tri-lineage (osteoblast, adipocyte, and chondrocyte) and suppress inflammation, are promising tools for regenerative medicine. MSCs are phenotypically diverse based on their tissue origins. However, the mechanisms underlying cell-type-specific gene expression patterns are not fully understood due to the lack of suitable strategy to identify the diversity. In this study, we investigated gene expression programs and chromatin accessibilities of MSCs by whole-transcriptome RNA-seq analysis and an assay for transposase-accessible chromatin using sequencing (ATAC-seq). We isolated MSCs from four tissues (femoral and vertebral bone marrow, adipose tissue, and lung) and analysed their molecular signatures. RNA-seq identified the expression of MSC markers and both RNA-seq and ATAC-seq successfully clustered the MSCs based on their tissue origins. Interestingly, clustering based on tissue origin was more accurate with chromatin accessibility signatures than with transcriptome profiles. Furthermore, we identified transcription factors potentially involved in establishing cell-type specific chromatin structures. Thus, epigenome analysis is useful to analyse MSC identity and can be utilized to characterize these cells for clinical use.

Aim In patients with diabetes or ischemia, angiogenesis and infection control are required for chronic leg ulcers, which substantially impair patients’ quality of life. We developed a novel functional peptide, named AG30/5C, with angiogenic and anti-microbial properties. Treatment with AG30/5C significantly accelerated the wound healing of full-thickness defects in mice. To evaluate the safety of AG30/5C in the treatment of leg ulcers, a physician-initiated clinical study was carried out. Methods The first-in-human trial was designed as an open-label treatment with AG30/5C (0.1 mg/mL) given twice per day for 11 days, and with a follow-up period of 17 days. The inclusion criteria for severe skin ulcers were: (i) diabetes or critical limb ischemia; (ii) resistance to standard therapy for 1 month; and (iii) detection of methicillin-resistant Staphylococcus aureus in the skin ulcer. Results Four patients were enrolled in this study, and two patients met these criteria. For the evaluation of safety, three adverse effects were reported as possibly related to AG30/5C treatment; however, these adverse effects were not severe and resolved during or after treatment. Thus, there were no safety concerns. In both patients, the size of the ulcer decreased after treatment (44.62% and 10.23% decrease), and further decreased after the follow-up period (73.85% and 10.23% decrease). The former patient was diagnosed as Werner syndrome and the skin ulcer was resistant to standard therapy; however, it was sensitive to AG30/5C treatment. Conclusions Topical treatment with AG30/5C for severe leg ulcers was safe, well tolerated and effective. Geriatr Gerontol Int 2017; ••: ••–••.

Background: Several new genes and clinical subtypes have been identified since the publication in 2014 of the report of the last International Consensus Meeting on Epidermolysis Bullosa (EB). Objectives: We sought to reclassify disorders with skin fragility, with a focus on EB, based on new clinical and molecular data. Methods: This was a consensus expert review. Results: In this latest consensus report, we introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Other disorders with skin fragility, where blisters are a minor part of the clinical picture or are not seen because skin cleavage is very superficial, are classified as separate categories. These include peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility. Because of the common manifestation of skin fragility, these ‘EB-related’ disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Conclusions: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and genetic features of EB. What is already known about this topic?. Epidermolysis bullosa (EB) is a group of genetic disorders with skin blistering. The last updated recommendations on diagnosis and classification were published in 2014. What does this study add?. We introduce the concept of genetic disorders with skin fragility, of which classical EB represents the prototype. Clinical and genetic aspects, genotype–phenotype correlations, disease-modifying factors and natural history of EB are reviewed. Other disorders with skin fragility, e.g. peeling skin disorders, erosive disorders, hyperkeratotic disorders, and connective tissue disorders with skin fragility are classified as separate categories; these ‘EB-related’ disorders should be considered under the EB umbrella in terms of medical and socioeconomic provision of care. Linked Comment: Pope. Br J Dermatol 2020; 183:603.

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a severe systemic genodermatosis lacking therapies beyond supportive care for its extensive, life-limiting manifestations. Objectives: To report the safety and preliminary responses of 10 patients with RDEB to bone marrow transplant (BMT) with post-transplant cyclophosphamide (PTCy BMT) after reduced-intensity conditioning with infusions of immunomodulatory donor-derived mesenchymal stromal cells (median follow-up 16 months). Methods: BMT toxicities, donor blood and skin engraftment, skin biopsies, photographic and dynamic assessments of RDEB disease activity were obtained at intervals from pre-BMT to 1 year post-BMT. Results: Related donors varied from haploidentical (n = 6) to human leucocyte antigen (HLA)-matched (n = 3), with one HLA-matched unrelated donor. Transplant complications included graft failure (n = 3; two pursued a second PTCy BMT), veno-occlusive disease (n = 2), posterior reversible encephalopathy (n = 1) and chronic graft-versus-host disease (n = 1; this patient died). In the nine ultimately engrafted patients, median donor chimerism at 180 days after transplant was 100% in peripheral blood and 27% in skin. Skin biopsies showed stable (n = 7) to improved (n = 2) type VII collagen protein expression by immunofluorescence and gain of anchoring fibril components (n = 3) by transmission electron microscopy. Early signs of clinical response include trends toward reduced body surface area of blisters/erosions from a median of 49·5% to 27·5% at 100 days after BMT (P = 0·05), with parental measures indicating stable quality of life. Conclusions: PTCy BMT in RDEB provides a means of attaining immunotolerance for future donor-derived cellular grafts (ClinicalTrials.gov identifier NCT02582775). What's already known about this topic?. Severe, generalized recessive dystrophic epidermolysis bullosa (RDEB) is marked by great morbidity and early death. No cure currently exists for RDEB. Bone marrow transplant (BMT) is the only described systemic therapy for RDEB. What does this study add?. The first description of post-transplant cyclophosphamide (PTCy) BMT for RDEB. PTCy was well tolerated and provided excellent graft-versus-host disease prophylaxis, replacing long courses of calcineurin inhibitors in patients receiving human leucocyte antigen-matched sibling BMT. What is the translational message?. The PTCy BMT platform permits identification of a suitable related donor for most patients and for subsequent adoptive transfer of donor nonhaematopoietic cells after establishment of immunological tolerance.

Objectives High-mobility group box 1 protein (HMGB1) fragment enhances bone marrow-derived mesenchymal stem cell (BM-MSC) recruitment to damaged tissue to promote tissue regeneration. This study aimed to evaluate whether systemic injection of HMGB1 fragment could promote tissue repair in a rat model of myocardial infarction (MI). Methods HMGB1 (n = 14) or phosphate buffered saline (n = 12, control) was administered to MI rats for 4 days. Cardiac performance and left ventricular remodeling were evaluated using ultrasonography and immunostaining. BM-MSC recruitment to damaged tissue in green fluorescent protein-bone marrow transplantation (GFP-BMT) models was evaluated using immunostaining. Results At four weeks post-treatment, the left ventricular ejection fraction was significantly improved in the HMGB1 group compared to that in the control. Interstitial fibrosis and cardiomyocyte hypertrophy were also significantly attenuated in the HMGB1 group compared to the control. In the peri-infarction area, VEGF-A mRNA expression was significantly higher and TGFβ expression was significantly attenuated in the HMGB1 group than in the control. In GFP-BMT rats, GFP+/PDGFRα+ cells were significantly mobilized to the peri-infarction area in the HMGB1 group compared to that in the control, leading to the formation of new vasculature. In addition, intravital imaging revealed that more GFP+/PDGFRα+ cells were recruited to the peri-infarction area in the HMGB1 group than in the control 12 h after treatment. Conclusions Systemic administration of HMGB1 induced angiogenesis and reduced fibrosis by recruiting PDGFRα+ mesenchymal cells from the bone marrow, suggesting that HMGB1 administration might be a new therapeutic approach for heart failure after MI.

Background High-mobility group box 1 protein (HMGB1) reportedly enhances CXCR4-positive bone marrow-derived mesenchymal stem cell (BM-MSC) recruitment to damaged tissue to promote tissue regeneration. Purpose Our aim of this study is to evaluate whether systemic administration of HMGB1 might promote tissue repair in a rat myocardial infarction (MI) model. Methods We prepared 26 MI model rats with high ligation of the left coronary artery. Two weeks later, HMGB1 (3 mg/kg/day) or phosphate-buffered saline (control: 3 mL/kg/day) was administered for 4 days via femoral vein. Cardiac performance was evaluated by ultrasonography, left ventricular (LV) remodeling via immunostaining. We then used immunostaining to examine MSC recruitment to damaged tissue in green fluorescent protein bone marrow transplantation (GFP-BMT) model rats, and also performed intravital imaging using two-photon microscopy to visualize BM-cells recruitment in real time. Results Compared with control rats, there was a significant improvement in the left ventricular ejection fraction of the HMGB1 group (HMGB1 vs. control: 48.6% ± 5.5% vs. 33.6% ± 5.4%; p Conclusions Systemic administration of HMGB1 mobilized BM-MSCs to the damaged myocardium via the SDF-1/CXCR4 signaling complex. Those BM-MSCs might migrate to extracellular matrix in the border zone via the gap of each endothelial cell, leading to induction of angiogenesis and reduced fibrosis. Acknowledgement/Funding None