Avaliação do papel desempenhado por vesículas extracelulares derivadas de glioblastoma na modulação do sistema imune e na progressão tumoral

O glioblastoma (GBM) é o mais maligno e com a menor taxa de sobrevida dos tumores gliais. O tecido tumoral é composto por muitas células neoplásicas em proliferação, fibroblastos e células do sistema imune. A proliferação tumoral depende de uma rede complexa de fatores, como citocinas, adenosina e vesículas extracelulares (VEs). O papel das VEs ainda é um assunto controverso e sua atividade pró-tumoral ou antitumoral não é totalmente compreendida. Neste contexto, o objetivo desse trabalho foi compreender o papel das vesículas extracelulares derivadas de tumores (VETs), derivadas da linhagem celular C6, na modulação do sistema imune e na progressão do GBM. VETs foram isolados por centrifugação diferencial do sobrenadante da linhagem celular C6. O tamanho e a polidispersão foram analisados por equipamentos Nanosight, ZetaSizer e Microscopia Eletrônica de Transmissão. A estabilidade das VETs foi analisada pelo equipamento ZetaSizer nos dias 1, 4 e 18. Estas vesículas foram caracterizadas pela presença de marcador clássico, como CD9, e também pela presença das enzimas CD39 e CD73. Além disso, as células de GBM C6 foram tratadas com diferentes concentrações de VETs durante 96 h (n = 3) e a viabilidade celular foi avaliada por ensaio MTS. Em seguida, as VETs foram incubadas (8 μg) com linfócitos mesentéricos isolados de ratos Wistar adultos. Após 48 h de incubação, a expressão das enzimas CD39 e CD73 nos linfócitos foi avaliada por citometria de fluxo. Os modelos GBM in vivo foram realizados com três grupos de ratos Wistar machos adultos: Controle, coinjeção e imunização. No grupo de coinjeção, as VETs foram coinjetadas com células C6 GBM no estriado por cirurgia estereotáxica. No grupo de imunização, os ratos foram tratados com 20 μg de VETs 10 e 5 dias antes da cirurgia. Após 14 dias de crescimento tumoral, os ratos foram decapitados e o cérebro foi removido para quantificação do volume do tumor e análise do microambiente tumoral. As VETs apresentaram tamanho uniforme (175,2 ± 6,14nm) e a estabilidade, a 4 ° C, durante os 18 dias testados. Os inibidores das enzimas CD39 e CD73 reduziram (52,1% e 57,8%, respectivamente) a formação de ADO, enquanto o efeito do bloqueio do receptor ADO não alterou a concentração deste nucleosídeo. A porcentagem de células viáveis foi significativamente reduzida após o tratamento com 16 e 32 μg / mL de VETs (de 120 ± 2,12% para 82,52 ± 5% e 92,1 ± 7,9%, respectivamente). A incubação de linfócitos T com VETs não alterou a expressão da proteína CD39 e CD73 em nenhum dos subtipos testado de linfócitos T. Além disso, a coinjeção de VETs reduziu o tamanho do GBM de 221 ± 65,1 mm³ para 121 ± 40,6 mm³ em comparação ao controle. O grupo imunização apresentou um menor volume de GBM em comparação ao grupo controle (de 173 ± 91,8 mm³ para 69 ± 20,2 mm³). Buscando entender o mecanismo por trás dessa redução, analisamos a presença de células CD4+FOXP3- e CD4+FOXP3+ no microambiente tumoral. O grupo coinjeção mostrou uma redução significativa de células CD4+FOXP3- e CD4+FOXP3+, no entanto, não observamos o mesmo no grupo de imunização, que não mostrou diferença entre os grupos. Juntos, nossos resultados sugerem que os VETs desempenham um papel antitumoral na progressão do GBM, reduzindo a proliferação tumoral e a presença de linfócitos T regulatórios no microambiente tumoral.Glioblastoma (GBM) is the most malignant with the poorest survival rate of the glial tumors. The tumoral tissue is composed by many proliferating neoplastic cells, fibroblasts and cells of immune system. Tumor proliferation depends on complex network factors, such as cytokines, adenosine and extracellular vesicles (EVs). The role of EVs remains controversial and their pro-tumoral or antitumoral activity is not fully understand. In this context, the aim of this study was understand the role of tumor-derived extracellular vesicles (TEVs) in the immune system modulation and GBM progression. TEVs were isolated by differential centrifugation of C6 cell line supernatant. Size and polydispersity were analyzed by Nanosight, Zetasizer equipments and Transmission Electron Microscopy. TEVs stability was analyzed by Zetasizer equipment on days 1, 4 and 18. These vesicles were characterized by the presence of EVs classical marker, CD9, also CD39 and CD73 enzymes. Further, C6 GBM cells were treated with different concentrations of TEVs during 96 h (n=3) and cell viability was assessed by MTS assay. Then, GEVs were incubated (8 μg) with mesenteric lymphocytes isolated from adult Wistar rats. After 48 h of incubation, the expression of CD39 and CD73 enzymes was evaluated by flow cytometry. The in vivo GBM model was performed with three adult male Wistar rats groups: Control, coinjection and immunization. In the coinjection group, TEVs were coinjecting with C6 GBM cells into the striatum by stereotactic surgery of adult Wistar rats. In the immunization group, rats were treated with 20 μg of TEVs 10 and 5 days before surgery. After 14 days of tumor growth, the rats were decapitated and the entire brain was removed for tumor size quantification and tumor microenvironment analysis. TEVs presented uniform size (175,2±6,14nm) and the stability, at 4°C, during the 18 days tested (186,8±6,64nm). Inhibitors of CD39 and CD73 enzymes reduces (52,1% and 57,8%, respectively) formation of ADO while the effect of ADO receptor blockade did not alter the concentration of this nucleoside. The percentage of viable cells was significantly reduced after treatment with 16 and 32 μg/mL of TEVs (from 120±2,12% to 82,52±5% and 92,1±7,9%, respectively). The incubation of T-lymphocytes with TEVs did not alter the expression of CD39 and CD73 protein in any tested subset of T-lymphocytes. Moreover, the co-injection of TEVs reduces the GBM size from 221 ± 65,1 mm³ to 121± 40,6 mm in comparison to GBM group. Immunization group reduces the GBM size from 173 ± 91,8 mm³ to 69 ± 20,2 mm³. Seeking to understand the mechanism behind this reduction, we analyzed the presence of CD4+FOXP3- and CD4+FOXP3+ cells in tumor microenvironment. The coinjection group showed a significant reduction of CD4+FOXP3- and CD4+ FOXP3+ cells, however we didn’t observe the same in immunization group, which has shown no difference between groups. Together, our results suggest GEVs plays an anti-tumoral role in GBM progression by reducing tumor proliferation and presence of T regulatory lymphocytes in tumor microenvironment

Nanoformulation shows cytotoxicity against glioblastoma cell lines and antiangiogenic activity in chicken chorioallantoic membrane

Glioblastoma (GB) is a histological and genetically heterogeneous brain tumor that is highly proliferative and vascularized. The prognosis is poor with currently available treatment. In this study, we evaluated the cytotoxicity and antiangiogenic activity of doxorubicin-loaded-chitosan-coated-arginylglycylaspartic acid-functionalized-poly(ε-caprolactone)-alpha bisabolol-LNC (AB-DOX-LNC-L-C-RGD). The nanoformulation was prepared by self-assembling followed by interfacial reactions, physicochemically characterized and evaluated in vitro against GB cell lines (U87MG and U138MG) and in vivo using the chicken chorioallantoic membrane assay (CAM). Spherical shape nanocapsules had a hydrodynamic mean diameter of 138 nm, zeta potential of +13.4 mV, doxorubicin encapsulation of 65%, and RGD conjugation of 92%. After 24 h of treatment (U87MG and U138MG), the median inhibition concentrations (IC50) were 520 and 490 nmol L−1 doxorubicin-equivalent concentrations, respectively. The treatment induced antiproliferative activity with S-phase cell-cycle arrest and apoptosis in the GB cells. Furthermore, after 48 h of exposure, evaluation of antiangiogenic activity (CAM) showed that the relative vessel growth following treatment with the nanocapsules was 5.4 times lower than that with the control treatment. The results support the therapeutic potential of the nanoformulation against GB and, thereby, pave the way for future preclinical studies

Lymphocytes from B-acute lymphoblastic leukemia patients present diferential regulation of the adenosinergic axis depending on risk stratifcation

Purpose Although risk-stratifed chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a signifcant number of cases. The identifcation of new therapeutic targets as well as prog nostic and diagnostic biomarkers can improve B-ALL patients’ clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients’ lymphocyte subpopulations. Methods Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of CD38+∕CD73+, and CD39+∕CD73+ in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). Results Comparing B-ALL patients to health donors, we observed an increase of CD4+and CD8+T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1β, and ADO concentrations, together with an increase in AMP in B-ALL patients’ plasma. Conclusion: As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of diferent cellular subsets. We observed a pro-tumor immunity expression profle in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL

Obesity drives adipose-derived stem cells into a senescent and dysfunctional phenotype associated with P38MAPK/NF-KB axis

Background: Adipose-derived stem cells (ADSC) are multipotent cells implicated in tissue homeostasis. Obesity represents a chronic inflammatory disease associated with metabolic dysfunction and age-related mechanisms, with progressive accumulation of senescent cells and compromised ADSC function. In this study, we aimed to explore mechanisms associated with the inflammatory environment present in obesity in modulating ADSC to a senescent phenotype. We evaluated phenotypic and functional alterations through 18 days of treatment. ADSC were cultivated with a conditioned medium supplemented with a pool of plasma from eutrophic individuals (PE, n = 15) or with obesity (PO, n = 14), and compared to the control. Results: Our results showed that PO-treated ADSC exhibited decreased proliferative capacity with G2/M cycle arrest and CDKN1A (p21WAF1/Cip1) up-regulation. We also observed increased senescence-associated β-galactosidase (SA-β-gal) activity, which was positively correlated with TRF1 protein expression. After 18 days, ADSC treated with PO showed augmented CDKN2A (p16INK4A) expression, which was accompanied by a cumulative nuclear enlargement. After 10 days, ADSC treated with PO showed an increase in NF-κB phosphorylation, while PE and PO showed an increase in p38MAPK activation. PE and PO treatment also induced an increase in senescence-associated secretory phenotype (SASP) cytokines IL-6 and IL-8. PO-treated cells exhibited decreased metabolic activity, reduced oxygen consumption related to basal respiration, increased mitochondrial depolarization and biomass, and mitochondrial network remodeling, with no superoxide overproduction. Finally, we observed an accumulation of lipid droplets in PO-treated ADSC, implying an adaptive cellular mechanism induced by the obesogenic stimuli. Conclusions: Taken together, our data suggest that the inflammatory environment observed in obesity induces a senescent phenotype associated with p38MAPK/NF-κB axis, which stimulates and amplifies the SASP and is associated with impaired mitochondrial homeostasis

Dysfunctional purinergic signaling correlates with disease severity in COVID-19 patients

Ectonucleotidases modulate inflammatory responses by balancing extracellular ATP and adenosine (ADO) and might be involved in COVID-19 immunopathogenesis. Here, we explored the contribution of extracellular nucleotide metabolism to COVID-19 severity in mild and severe cases of the disease. We verified that the gene expression of ectonucleotidases is reduced in the whole blood of patients with COVID-19 and is negatively correlated to levels of CRP, an inflammatory marker of disease severity. In line with these findings, COVID-19 patients present higher ATP levels in plasma and reduced levels of ADO when compared to healthy controls. Cell type-specific analysis revealed higher frequencies of CD39+ T cells in severely ill patients, while CD4+ and CD8+ expressing CD73 are reduced in this same group. The frequency of B cells CD39+CD73+ is also decreased during acute COVID-19. Interestingly, B cells from COVID-19 patients showed a reduced capacity to hydrolyze ATP into ADP and ADO. Furthermore, impaired expression of ADO receptors and a compromised activation of its signaling pathway is observed in COVID-19 patients. The presence of ADO in vitro, however, suppressed inflammatory responses triggered in patients’ cells. In summary, our findings support the idea that alterations in the metabolism of extracellular purines contribute to immune dysregulation during COVID-19, possibly favoring disease severity, and suggest that ADO may be a therapeutic approach for the disease

Avaliação do papel desempenhado por vesículas extracelulares derivadas de glioblastoma na modulação do sistema imune e na progressão tumoral

O glioblastoma (GBM) é o mais maligno e com a menor taxa de sobrevida dos tumores gliais. O tecido tumoral é composto por muitas células neoplásicas em proliferação, fibroblastos e células do sistema imune. A proliferação tumoral depende de uma rede complexa de fatores, como citocinas, adenosina e vesículas extracelulares (VEs). O papel das VEs ainda é um assunto controverso e sua atividade pró-tumoral ou antitumoral não é totalmente compreendida. Neste contexto, o objetivo desse trabalho foi compreender o papel das vesículas extracelulares derivadas de tumores (VETs), derivadas da linhagem celular C6, na modulação do sistema imune e na progressão do GBM. VETs foram isolados por centrifugação diferencial do sobrenadante da linhagem celular C6. O tamanho e a polidispersão foram analisados por equipamentos Nanosight, ZetaSizer e Microscopia Eletrônica de Transmissão. A estabilidade das VETs foi analisada pelo equipamento ZetaSizer nos dias 1, 4 e 18. Estas vesículas foram caracterizadas pela presença de marcador clássico, como CD9, e também pela presença das enzimas CD39 e CD73. Além disso, as células de GBM C6 foram tratadas com diferentes concentrações de VETs durante 96 h (n = 3) e a viabilidade celular foi avaliada por ensaio MTS. Em seguida, as VETs foram incubadas (8 μg) com linfócitos mesentéricos isolados de ratos Wistar adultos. Após 48 h de incubação, a expressão das enzimas CD39 e CD73 nos linfócitos foi avaliada por citometria de fluxo. Os modelos GBM in vivo foram realizados com três grupos de ratos Wistar machos adultos: Controle, coinjeção e imunização. No grupo de coinjeção, as VETs foram coinjetadas com células C6 GBM no estriado por cirurgia estereotáxica. No grupo de imunização, os ratos foram tratados com 20 μg de VETs 10 e 5 dias antes da cirurgia. Após 14 dias de crescimento tumoral, os ratos foram decapitados e o cérebro foi removido para quantificação do volume do tumor e análise do microambiente tumoral. As VETs apresentaram tamanho uniforme (175,2 ± 6,14nm) e a estabilidade, a 4 ° C, durante os 18 dias testados. Os inibidores das enzimas CD39 e CD73 reduziram (52,1% e 57,8%, respectivamente) a formação de ADO, enquanto o efeito do bloqueio do receptor ADO não alterou a concentração deste nucleosídeo. A porcentagem de células viáveis foi significativamente reduzida após o tratamento com 16 e 32 μg / mL de VETs (de 120 ± 2,12% para 82,52 ± 5% e 92,1 ± 7,9%, respectivamente). A incubação de linfócitos T com VETs não alterou a expressão da proteína CD39 e CD73 em nenhum dos subtipos testado de linfócitos T. Além disso, a coinjeção de VETs reduziu o tamanho do GBM de 221 ± 65,1 mm³ para 121 ± 40,6 mm³ em comparação ao controle. O grupo imunização apresentou um menor volume de GBM em comparação ao grupo controle (de 173 ± 91,8 mm³ para 69 ± 20,2 mm³). Buscando entender o mecanismo por trás dessa redução, analisamos a presença de células CD4+FOXP3- e CD4+FOXP3+ no microambiente tumoral. O grupo coinjeção mostrou uma redução significativa de células CD4+FOXP3- e CD4+FOXP3+, no entanto, não observamos o mesmo no grupo de imunização, que não mostrou diferença entre os grupos. Juntos, nossos resultados sugerem que os VETs desempenham um papel antitumoral na progressão do GBM, reduzindo a proliferação tumoral e a presença de linfócitos T regulatórios no microambiente tumoral.Glioblastoma (GBM) is the most malignant with the poorest survival rate of the glial tumors. The tumoral tissue is composed by many proliferating neoplastic cells, fibroblasts and cells of immune system. Tumor proliferation depends on complex network factors, such as cytokines, adenosine and extracellular vesicles (EVs). The role of EVs remains controversial and their pro-tumoral or antitumoral activity is not fully understand. In this context, the aim of this study was understand the role of tumor-derived extracellular vesicles (TEVs) in the immune system modulation and GBM progression. TEVs were isolated by differential centrifugation of C6 cell line supernatant. Size and polydispersity were analyzed by Nanosight, Zetasizer equipments and Transmission Electron Microscopy. TEVs stability was analyzed by Zetasizer equipment on days 1, 4 and 18. These vesicles were characterized by the presence of EVs classical marker, CD9, also CD39 and CD73 enzymes. Further, C6 GBM cells were treated with different concentrations of TEVs during 96 h (n=3) and cell viability was assessed by MTS assay. Then, GEVs were incubated (8 μg) with mesenteric lymphocytes isolated from adult Wistar rats. After 48 h of incubation, the expression of CD39 and CD73 enzymes was evaluated by flow cytometry. The in vivo GBM model was performed with three adult male Wistar rats groups: Control, coinjection and immunization. In the coinjection group, TEVs were coinjecting with C6 GBM cells into the striatum by stereotactic surgery of adult Wistar rats. In the immunization group, rats were treated with 20 μg of TEVs 10 and 5 days before surgery. After 14 days of tumor growth, the rats were decapitated and the entire brain was removed for tumor size quantification and tumor microenvironment analysis. TEVs presented uniform size (175,2±6,14nm) and the stability, at 4°C, during the 18 days tested (186,8±6,64nm). Inhibitors of CD39 and CD73 enzymes reduces (52,1% and 57,8%, respectively) formation of ADO while the effect of ADO receptor blockade did not alter the concentration of this nucleoside. The percentage of viable cells was significantly reduced after treatment with 16 and 32 μg/mL of TEVs (from 120±2,12% to 82,52±5% and 92,1±7,9%, respectively). The incubation of T-lymphocytes with TEVs did not alter the expression of CD39 and CD73 protein in any tested subset of T-lymphocytes. Moreover, the co-injection of TEVs reduces the GBM size from 221 ± 65,1 mm³ to 121± 40,6 mm in comparison to GBM group. Immunization group reduces the GBM size from 173 ± 91,8 mm³ to 69 ± 20,2 mm³. Seeking to understand the mechanism behind this reduction, we analyzed the presence of CD4+FOXP3- and CD4+FOXP3+ cells in tumor microenvironment. The coinjection group showed a significant reduction of CD4+FOXP3- and CD4+ FOXP3+ cells, however we didn’t observe the same in immunization group, which has shown no difference between groups. Together, our results suggest GEVs plays an anti-tumoral role in GBM progression by reducing tumor proliferation and presence of T regulatory lymphocytes in tumor microenvironment

Inhibition of ATP hydrolysis as a key regulator of temozolomide resistance and migratory phenotype of glioblastoma cells

Glioblastoma (GBM) is the most lethal among malignant gliomas. The tumor invasiveness and therapy-resistance are important clinical hallmarks. Growing evidence emphasizes the purinergic signaling contributing to tumor growth. Here we exposed a potential role of extracellular ATPase activity as a key regulator of temozolomide cytotoxicity and the migration process in GBM cells. The inhibition of ATP hydrolysis was able to improve the impact of temozolomide, causing arrest mainly in S and G2 phases of the cell cycle, leading M059J and U251 cells to apoptosis. In addition to eradicating GBM cells, ATP hydrolysis exhibited a potential to modulate the invasive phenotype and the expression of proteins involved in cell migration and epithelial-to-mesenchymal-like transition in a 3D culture model. Finally, we suggest the ATPase activity as a key target to decline temozolomide resistance and the migratory phenotype in GBM cells

3-O-Methylquercetin from Achyrocline satureioides—cytotoxic activity against A375-derived human melanoma cell lines and its incorporation into cyclodextrins-hydrogels for topical administration

3-O-Methylquercetin (3OMQ), a natural 3-O-methylflavonoid, was isolated from Achyrocline satureioides and purified using the high-performance counter current chromatography (HPCCC) on a semi-preparative scale. High-purity 3OMQ (98%) was obtained with excellent recovery (81.8% (w/w)) and good yield (190 mg/100 g of plant). Isolated 3OMQ was evaluated against the A375 human amelanotic melanoma cancer cell line and A375-derived with different degrees of aggressiveness (A375-A7, A375-G10, and A375-PCDNA3). The results showed that 3OMQ reduced the cell viability of all strains, demonstrating time- and dose-dependent responses. 3OMQ was used to obtain hydrogels for the topical treatment of melanoma. Thus, 3OMQ was incorporated into hypromellose hydrogels with/without different cyclodextrins (CDs). The 3OMQ formulations showed permeation/retention in all skin layers, namely stratum corneum, epidermis, and dermis. A significant amount of 3OMQ was found in the replication site of the melanoma cells (epidermis and dermis). Altogether, these results demonstrate that 3OMQ can be isolated from Achyrocline satureioides by HPCCC on a semi-preparative scale and exhibit cytotoxic activity against melanoma cells. Its incorporation into an HPMC hydrogel containing HP-β-CD yielded a formulation with excellent technological and biopharmaceutical characteristics for evaluating the topical management of melanoma.Fil: Doneda, Eduarda. Universidade Federal do Rio Grande do Sul; BrasilFil: Bianchi, Sara Elis. Universidade Federal do Rio Grande do Sul; BrasilFil: Pittol, Vanessa. Universidade Federal do Rio Grande do Sul; BrasilFil: Kreutz, Tainá. Universidade Federal do Rio Grande do Sul; BrasilFil: Scholl, Juliete Nathali. Universidade Federal do Rio Grande do Sul. Instituto de Ciências Básicas da Saude; BrasilFil: Ibañez, Irene Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche | Comisión Nacional de Energía Atómica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche; ArgentinaFil: Bracalente, María Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche | Comisión Nacional de Energía Atómica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche; ArgentinaFil: Duran, Hebe Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche | Comisión Nacional de Energía Atómica. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología. Unidad Ejecutora Instituto de Nanociencia y Nanotecnología - Nodo Bariloche; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Figueiró, Fabrício. Universidade Federal do Rio Grande do Sul. Instituto de Ciências Básicas da Saude; BrasilFil: Klamt, Fabio. Universidade Federal do Rio Grande do Sul. Instituto de Ciências Básicas da Saude; BrasilFil: Bassani, Valquiria Linck. Universidade Federal do Rio Grande do Sul; Brasi