IGF1受体的β亚基突变体对人骨肉瘤细胞增殖、迁移和凋亡的影响

目的 探讨IGF1受体（IGF-1R）的β亚基突变体（sb-IGF1R、ma-IGF1R）对骨肉瘤143B细胞生物学行为的影响。方法 设计与构建sb-IGF1R、ma-IGF1R片段,克隆到腺病毒AdEasy穿梭质粒中,获得Ad-sbIGF1R、Ad-maIGF1R,再将两者与Ad-GFP分别转染骨肉瘤细胞143B,观察细胞的增殖、迁移及凋亡情况;构建Ad-sbIGF1R、Ad-maIGF1R及Ad-GFP裸鼠皮下荷瘤模型,评价肿瘤在体外的生长情况。结果 通过质粒PCR验证,IGF-1Rβ亚基突变体在骨肉瘤细胞中过表达。与对照组相比,经Ad-sbIGF1R、Ad-maIGF1R处理过的骨肉瘤细胞,其细胞增殖、迁移能力显著抑制,促进了骨肉瘤细胞的凋亡。同时在裸鼠皮下荷瘤模型中抑制肿瘤生长。结论 IGF1受体的β亚基突变体抑制骨肉瘤细胞增殖和转移,并促进骨肉瘤细胞凋亡。     

腺病毒介导的shRNA沉默hTERT基因表达对鼻咽癌细胞增殖和凋亡的影响

目的探讨靶向人端粒酶反转录酶（hTERT)的短发夹RNA（shRNA)对人鼻咽癌细胞株CNE-2 hTERT表达的影响,及其对鼻咽癌细胞增殖和凋亡的效应。方法构建表达绿色荧光蛋白(EGFP)基因和靶向hTERT基因短发夹RNA的重组腺病毒质粒,观察其对鼻咽癌细胞株(CNE-2)的转染效果,RT-PCR检测hTERT mRNA表达水平,Western blot检测hTERT蛋白表达水平,CCK-8法检测细胞增殖活性,流式细胞仪检测细胞凋亡状况。结果Adv-EGFP-shTERT重组腺病毒质粒转染率可达90%以上,成功转染CNE-2细胞24 h后,hTERT mRNA的表达水平显著下降,转染48 h后,hTERT蛋白表达明显下调,细胞增殖活性受到显著抑制,细胞凋亡率可达23.0%。结论腺病毒载体介导靶向hTERT基因的RNA干扰,能显著抑制端粒酶反转录酶表达,进而抑制端粒酶活性,抑制CNE-2细胞增殖并诱导其凋亡,为鼻咽癌的基因治疗研究提供了理论基础。     

EPHA2-siRNA质粒对骨肉瘤细胞生物学行为的影响

目的探讨骨肉瘤细胞中EPHA2基因表达对细胞增殖、凋亡以及仿血管发生的影响。方法应用pSiliencer质粒构建靶向EPHA2的siRNA质粒;脂质体转染siRNA质粒人骨肉瘤细胞,Western blot在蛋白水平检测转染前后细胞基因表达情况;采用四甲基偶氮唑盐(MTT)法、流式细胞仪结合Annexin V-FITC和PI染色以及HE染色观察转染前后骨肉瘤细胞增殖、凋亡及仿血管发生情况。结果成功构建了针对EPHA2的siRNA表达质粒;将质粒转染入骨肉瘤后,EPHA2基因在蛋白表达水平下调;骨肉瘤细胞增殖能力下降,并发生了早期凋亡,骨肉瘤细胞的仿血管发生受到抑制。结论EPHA2参与了骨肉瘤细胞增殖、早期凋亡和仿血管发生;利用RNA干扰技术靶向EPHA2基因治疗可能为骨肉瘤治疗提供新方法。     

长链非编码RNA TUG1调控miR-138-5p对骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响

目的:探讨长链非编码RNA(LncRNA)牛磺酸调节基因1(TUG1)调控miR-138-5p对骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响。方法:在骨肉瘤细胞U-2OS中转染TUG1 siRNA和siRNA control,qRT-PCR测定干扰效果,MTT测定增殖,流式细胞术测定凋亡,Transwell小室测定细胞侵袭和迁移。starBase预测TUG1与miR-138-5p有结合位点,双荧光素酶报告载体鉴定靶向调控关系。将miR-138-5p抑制物和TUG1 siRNA共转染至骨肉瘤细胞中,测定干扰miR-138-5p对下调TUG1的骨肉瘤细胞增殖、凋亡、侵袭和迁移的影响。结果:转染TUG1 siRNA后的骨肉瘤细胞中TUG1表达水平明显低于转染siRNA control后的细胞（P<0.05）。下调TUG1后的骨肉瘤细胞增殖、侵袭和迁移能力降低,细胞凋亡率升高（P<0.05）。野生型TUG1荧光素酶报告载体与miR-138-5p共转染后细胞荧光素酶活性降低（P<0.05）。与转染TUG1 siRNA的细胞比较,miR-138-5p抑制物和TUG1 siRNA共转染后的细胞增殖、侵袭和迁移能力升高,细胞凋亡率降低（P<0.05）。结论:下调LncRNA TUG1表达通过调控miR-138-5p表达抑制骨肉瘤细胞增殖、侵袭、迁移能力并诱导细胞凋亡。     

凋亡抑制基因c-FLIPs对人肺腺癌细胞增殖和凋亡的作用

目的:探讨过表达凋亡抑制基因c-FLIPs重组腺病毒对人肺腺癌细胞增殖和凋亡的作用。方法:构建过表达凋亡抑制c-FLIPs的重组腺病毒Ad5 c-FLIPs,感染人肺腺癌细胞Calu-3。Real-time PCR检测感染前后c-FLIPs、Caspase-8,Caspase-10和Bcl-2的表达。MTT法检测细胞增殖。流式细胞仪检测感染Ad5 c-FLIPs后人肺腺癌细胞的凋亡情况。结果:成功构建过表达c-FLIPs重组腺病毒Ad5 c-FLIPs,real-time PCR检测凋亡抑制基因c-FLIPs在Calu-3细胞株高表达,过表达c-FLIPs基因,凋亡相关基因Caspase-8、Caspase-10和Bcl-2的表达明显降低。MTT法检测感染前后细胞增殖情况发现,过表达c-FLIPs基因可诱导细胞增殖,流式细胞仪分析经PI染色后的Calu-3细胞株,对照组和腺病毒感染组细胞的凋亡率分别为（55.17±9.68）%和（10.97±1.66）%,差异具有统计学意义（P<0.05）。结论:凋亡抑制基因c-FLIPs可明显诱导人肺腺癌细胞增殖并抑制凋亡。     

腺苷抑制乳腺癌细胞增殖和迁移的机制

目的:检测腺苷对乳腺肿瘤细胞增殖及迁移能力的影响,并探究其潜在的分子机制。方法:以MCF-7细胞为模型,采用梯度浓度的腺苷处理细胞,MTT及细胞计数法检测腺苷对细胞增殖的影响,流式细胞术检测腺苷对细胞凋亡及周期的影响,划痕修复实验检测腺苷对细胞迁移能力的影响。结果:腺苷可显著抑制MCF-7细胞增殖,且抑制效果与腺苷浓度及作用时间呈相关性。流式细胞术检测结果表明腺苷可诱导MCF-7细胞凋亡,并引起细胞的 G2/M期阻滞。划痕实验结果表明腺苷抑制MCF-7细胞的迁移能力。实时定量PCR检测结果发现,促细胞凋亡分子Bax及Bak表达量显著升高,而凋亡抑制分子Bcl-2表达量显著下降,同时Caspase-3的表达量也显著升高。结论:腺苷可诱导MCF-7细胞凋亡,抑制细胞的增殖,同时腺苷可以抑制MCF-7细胞的迁移能力。腺苷诱导MCF-7细胞凋亡与其激活线粒体凋亡通路密切相关。     

CerS2对膀胱癌细胞增殖、迁移及AKT/mTOR信号通路的影响

目的:探究CerS2基因在膀胱癌细胞中的作用及其可能的机制。方法:构建pEGFP-C1-CerS2重组表达载体,并转染膀胱癌细胞系T24,使用蛋白质免疫印迹实验检测GFP-CerS2的表达;运用CCK-8法检测细胞增殖活性;运用Annexin Ｖ荧光标记实验检测细胞凋亡水平;分别使用细胞划痕实验及Transwell实验检测细胞迁移能力和侵袭能力;使用蛋白质免疫印迹实验检测细胞中AKT、p-AKT、mTOR及p-mTOR的表达水平。结果:重组GFP-CerS2融合蛋白在T24细胞中可以正常表达,过表达GFP-CerS2显著抑制了膀胱癌细胞的增殖活性,且显著增加了细胞凋亡水平。过表达GFP-CerS2抑制了膀胱癌细胞的迁移和侵袭能力。此外,过表达GFP-CerS2也显著抑制了膀胱癌细胞中AKT和mTOR的磷酸化修饰,但不影响总AKT及mTOR的表达水平。结论:过表达CerS2可以抑制AKT/mTOR信号通路,影响膀胱癌细胞的增殖和凋亡,并抑制细胞的迁移和侵袭能力。     

沉默SEMA4D对骨肉瘤细胞增殖、侵袭、凋亡的影响及机制

目的:研究沉默SEMA4D对骨肉瘤细胞增殖、侵袭和凋亡的影响,并探索其作用机制。方法:体外培养人骨肉瘤HOS和U2OS细胞并转染SEMA4D-siRNA（si-SEMA4D）。采用CCK-8、Annexin-V/PI双染、Caspase 3/7活性细胞凋亡、Transwell实验检测si-SEMA4D对细胞增殖、凋亡和侵袭等的影响。结果:SEMA4D在骨肉瘤组织和细胞中高表达。转染si-SEMA4D可有效地抑制HOS和U2OS细胞中SEMA4D的表达,同时转染si-SEMA4D显著降低了HOS和U2OS细胞的增殖能力和侵袭能力,诱导了HOS和U2OS细胞的凋亡。此外,转染si-SEMA4D显著抑制了MMP2/9、RhoA、ROCK1/2的表达。结论:SEMA4D在骨肉瘤组织和细胞中表达上调。抑制SEMA4D可能通过调控RhoA-ROCK信号通路和MMP2/9的表达诱导骨肉瘤细胞凋亡并抑制细胞增殖和侵袭。     

Livin促进骨肉瘤细胞增殖和侵袭作用的实验研究

目的：探讨凋亡抑制蛋白（IAP）家族成员Livin基因在骨肉瘤发生发展以及转移中的作用.方法：联合RNA干扰和质粒重组技术构建Livin基因短发夹RNA （shRNA）表达载体,筛选稳定沉默Livin表达的克隆.四甲基偶氮唑蓝法（MTT法）测定细胞增殖状况.划痕实验（Wound healing）和侵袭实验（Matrigel invasion assay）评价沉默Livin表达后细胞迁移和浸润能力的变化.免疫印迹法（Western blot）检测细胞周期蛋白Cyclin D1和基质金属蛋白酶-2,9（MMP-2,9）的蛋白表达变化.结果：shRNA稳定转染的骨肉瘤U2-OS细胞中Livin mRNA和蛋白水平明显下调.与对照组相比,细胞增殖,游走和侵袭能力明显下降,并且该克隆中Cyclin D1,MMP-2和MMP-9的蛋白表达也明显减弱.结论：凋亡抑制蛋白Livin能够在体外条件下促进骨肉瘤细胞增殖和侵袭作用,这一过程可能与Cyclin D1和MMP-2,9的表达增强有关.     

抑制Survivin基因对人卵巢癌SKOV3细胞增殖和凋亡的影响

目的:探讨应用RNAi技术沉默Survivin基因对人卵巢癌SKOV3细胞的影响。方法:构建Survivin基因shRNA真核表达载体,转染人卵巢癌SKOV3细胞。RT-PCR及Western blot检测Survivin基因的表达,MTT实验、流式细胞仪检测细胞增殖、凋亡的变化。结果:siRNA实验组细胞Survivin蛋白及mRNA表达水平明显下降,细胞增殖能力显著降低,细胞凋亡率显著升高。结论:应用RNAi技术沉默Survivin基因可以降低卵巢癌SKOV3细胞Survivin基因的表达,进而抑制肿瘤细胞的生长、增殖并诱导细胞凋亡。因此,Survivin基因可能成为抗肿瘤治疗的潜在靶点。     

A Salmonella Typhimurium mutant strain capable of RNAi delivery: Higher tumor-targeting and lower toxicity

Bacteria are highly versatile and useful tools that could deliver short interfering RNA. In this study, a phoP/phoQ double-deleted Salmonella Typhimurium named VNP(PhoP/Q⁻) based on the genetic background of VNP20009. The biological safety and function of VNP(PhoP/Q⁻) were also analyzed. Our study revealed the following results: (1) VNP(PhoP/Q⁻) exhibited lower titers in tumor-free livers and spleens than VNP20009, (2) The survival of VNP(PhoP/Q⁻) in macrophages and 4T1 tumor cells was significantly reduced compared with that of VNP20009, (3) The tumor-targeting ability of VNP(PhoP/Q⁻) was significantly enhanced compared with that of VNP20009, and the anticancer effects of VNP(pPhoP/Q⁻) and VNP20009 on tumor-bearing mice were similar, (4) VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid and express the EGFP reporter gene in tumor tissue. Therefore, VNP(PhoP/Q⁻) exhibited a better safety level in tumor-free mice and elicited an anti-tumor effect on tumor-bearing mice. Moreover, VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid into the cytoplasm of host cells to silence targeted genes.     

Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models

VNP20009, a genetically modified strain of Salmonella typhimurium with deletions in the msbB and purI loci, exhibited antitumor activities when given systemically to tumor-bearing mice. VNP20009 inhibited the growth of subcutaneously implanted B16F10 murine melanoma, and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA-MB-231. A single intravenous injection of VNP20009, at doses ranging from 1 x 10(4) to 3 x 10(6) cfu/mouse, produced tumor growth inhibitions of 57-95%. Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP20009. Using mice with immune system deficiencies, we also demonstrated that the antitumor effects of VNP20009 did not depend on the presence of T and B cells. In addition, VNP20009, given intravenously, inhibited the growth of lung metastases in mice. Only live bacteria showed the antitumor effect.     

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Salmonella-mediated tumor-targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.     

Synergistic effects of arsenic trioxide combined with Salmonella typhimurium in treating the advanced hepatocellular carcinoma in rat models

BackgroundTo evaluate the safety and efficacy of arsenic trioxide (ATO) combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 in treating the advanced hepatocellular carcinoma (HCC).MethodsThe proliferation assay, migration assay and real-time PCR analyses were performed to assess the impact of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 on the McA-RH7777 cells. Forty Buffalo rats were orthotopically implanted with HCC in the livers and randomly divided into four groups: (A) ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; (B) ATO; (C) {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; and (D) control. ATO (2 mg/kg) was administered by peritoneal injection once a day and continued for five days. {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 (about 1×10⁷ CFU) was directly injected into the tail vein. MRI examinations were performed to access the tumor responses one and 2 weeks later, respectively. Micro CT scans of chest were performed to assess the lung metastases. Hematoxylin-eosin (HE) staining and immunohistochemical analyses were performed to analyze the tumor tissues.ResultsIn the in vitro experiments, {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 suppressed the proliferation of McA-RH7777 cells, attenuated their migration ability, and weakened the potential of metastases. MRI examinations showed that the mean residual tumor volumes of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group on the 7th day and 14th day after the administration of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 were significantly smaller than those of other groups. Micro CT scans revealed that the lung metastases rates of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group and {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were significantly lower than those of other groups. Immunohistochemical analyses displayed that the levels of VEGF and Vimentin in the tumors of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were obviously lower than those of other groups. The median survival of rats in the ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group was longer than those of other groups.ConclusionsThe strategy of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 was safe and had a potential to inhibit tumor growth, decrease the lung metastases, and prolong the overall survival in treating the advanced HCC. The two complementary interventions may have synergistic effects.     

Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor targeting and virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in Phase I clinical trials, but is more attenuated than S. typhimurium A1-R and in addition of multiple amino-acid auxotrophs, is purine auxotropic with the purI mutation. In the present study, mouse Lewis lung carcinoma-bearing nude mouse models were treated with S. typhimurium A1-R or VNP20009. S. typhimurium A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via the tail vein. However, A1-R showed higher tumor targeting and inhibited the Lewis lung carcinoma to a greater extent than VNP20009, with less body weight loss. The mice tolerated S. typhimurium A1-R to at a least 2-fold higher dose than VNP20009 when the bacteria were administered iv. The results of the present study suggest that S. typhimurium A1-R has greater clinical potential than VNP20009.     

Suppression of pancreatic ductal adenocarcinoma growth by intratumoral delivery of attenuated Salmonella typhimurium using a dual fluorescent live tracking system

Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis among all malignancies and is resistant to almost all current therapies. Attenuated Salmonella typhimurium strain VNP20009 has been deployed as powerful anticancer agent in a variety of animal cancer models, and previous phase 1 clinical trials have proven its safety profiles. However, thus far, little is known about its effect on PDAC. Here, we established CFPAC-1 cell lines expressing an mKate2 protein and thus emitting far-red fluorescence in the subsequent xenograft implant. VNP20009 strain was further engineered to carry a luciferase cDNA, which catalyzes the light-emitting reaction to allow the observation of salmonella distribution and accumulation within tumor with live imaging. Using such VNP20009 strain and intratumoral delivery, we could reduce the growth of pancreatic cancer by inducing apoptosis and severe necrosis in a dosage dependent manner. Consistent with this finding, intratumoral delivery of VNP20009 also increase caspase-3 activity and the expression of Bax protein. In summary, we revealed that VNP20009 is a promising bacterial agent for the treatment of PDAC, and that we have established a dual fluorescent imaging system as a valuable tool for noninvasive live imaging of solid tumor and engineered bacterial drug.     

Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma

Activation of receptor tyrosine kinase (RTK) signalling pathways is frequently correlated to cancer cell proliferation, angiogenesis and cell survival. Sprouty (SPRY) proteins function as a physiological endogenous inhibitor of RTK signalling pathways, have been shown to be deregulated in most cancer cells. Here, we demonstrated that over-expression of SPRY1 and SPRY2 inhibited B16F10 cell proliferation through G1 phase arrest in vitro, and SPRY2 showed more potent inhibitory effects than SPRY1. In order to tumor-specific delivery of SPRY1/2 in vivo, two strains of attenuated Salmonella typhimurium VNP20009 (VNP-PQE-SPRY1 and VNP-PQE-SPRY2) were constructed to specifically express SPRY1 or SPRY2 under the control of a hypoxia-induced nirB promoter. The efficiency and specificity of the recombinant strains were validated in both bacteria and animal tumor models. SPRY1 and SPRY2 gene could be specifically driven by the nirB promoter under hypoxia, but not normoxia conditions. In addition, the tumor-targeting ability of VNP-PQE-SPRY1 or VNP-PQE-SPRY2 was similar with VNP. VNP-PQE-SPRY2 significantly suppressed melanoma growth in vivo, suggesting that SPRY2 is a more efficient agent for melanoma therapy. Moreover, the antitumor effect of VNP-SPRY2 is mainly mediated through the inhibition of ERK1/2 phosphorylation, which leads to the inhibition of proliferation in melanoma. Taken together, our results indicated that SPRY2 displayed more potent melanoma suppression than SPRY1 both in vitro and in vivo, and the hypoxia-induced tumor-specific gene therapy of SPRY2 delivered by VNP20009 is a promising strategy for melanoma therapy.