Nanoparticles for inducing Gaucher disease-like damage in cancer cells.

Nutrient avidity is one of the most distinctive features of tumours. However, nutrient deprivation has yielded limited clinical benefits. In Gaucher disease, an inherited metabolic disorder, cells produce cholesteryl-glucoside which accumulates in lysosomes and causes cell damage. Here we develop a nanoparticle (AbCholB) to emulate natural-lipoprotein-carried cholesterol and initiate Gaucher disease-like damage in cancer cells. AbCholB is composed of a phenylboronic-acid-modified cholesterol (CholB) and albumin. Cancer cells uptake the nanoparticles into lysosomes, where CholB reacts with glucose and generates a cholesteryl-glucoside-like structure that resists degradation and aggregates into microscale crystals, causing Gaucher disease-like damage in a glucose-dependent manner. In addition, the nutrient-sensing function of mTOR is suppressed. It is observed that normal cells escape severe damage due to their inferior ability to compete for nutrients compared with cancer cells. This work provides a bioinspired strategy to selectively impede the metabolic action of cancer cells by taking advantage of their nutrient avidity.

Chiral coordination polymer nanowires boost radiation-induced in situ tumor vaccination.

Radiation-induced in situ tumor vaccination alone is very weak and insufficient to elicit robust antitumor immune responses. In this work, we address this issue by developing chiral vidarabine monophosphate-gadolinium nanowires (aAGd-NWs) through coordination-driven self-assembly. We elucidate the mechanism of aAGd-NW assembly and characterize their distinct features, which include a negative surface charge, ultrafine topography, and right-handed chirality. Additionally, aAGd-NWs not only enhance X-ray deposition but also inhibit DNA repair, thereby enhancing radiation-induced in situ vaccination. Consequently, the in situ vaccination induced by aAGd-NWs sensitizes radiation enhances CD8+ T-cell-dependent antitumor immunity and synergistically potentiates the efficacy immune checkpoint blockade therapies against both primary and metastatic tumors. The well-established aAGd-NWs exhibit exceptional therapeutic capacity and biocompatibility, offering a promising avenue for the development of radioimmunotherapy approaches.

Temporal Asymmetry of Pleasant and Unpleasant Feelings Among Chinese Adolescents.

Previous studies have shown that anticipation induces more emotions than retrospection, known as temporal emotion asymmetry. However, the majority of previous studies have been confined to Western contexts. Eastern populations tend to emphasize the past more than their Western counterparts and may exhibit distinct forms of temporal emotion asymmetry. Therefore, we conducted an investigation involving Chinese adolescents. Our research encompassed two experiments, investigating Chinese adolescents' temporal emotion asymmetry from a self-perspective (Experiment 1; N = 124) and an other-perspective (Experiment 2; N = 162). Participants were prompted to retrospect and anticipate events that elicited pleasant or unpleasant feelings. The results revealed that, whether from a self-perspective or an other-perspective, retrospection of past positive events elicited greater pleasure than the anticipation of future positive events. However, concerning adverse events, under a self-perspective, anticipation induced more displeasure than retrospection (Experiment 1); under an other-perspective, retrospection induced more displeasure than anticipation (Experiment 2). Our findings provide some support for the construal level theory, fading affect bias, and mobilization-minimization hypothesis of event cognition. Based on these results, retrospection seems to be a potential means for regulating the emotions of Chinese adolescents.

Assessing NH300094, a novel dopamine and serotonin receptor modulator with cognitive enhancement property for treating schizophrenia.

Background: Schizophrenia is a serious psychiatric disorder that significantly affects the quality of life of patients. The objective of this study is to discover a novel antipsychotic candidate with highly antagonistic activity against both serotonin and dopamine receptors, demonstrating robust efficacy in animal models of positive, negative, and cognitive symptoms of schizophrenia. Methods: In the present study, we examined the activity of antipsychotic drug (NH300094) on 5-HT2A, 5-HT2C, 5-HT1A, 5-HT1B, 5-HT7, H1, M1, Alpha1A, D2L, D2S, Alpha2A, D3 receptor functional assay in vitro. In addition, multiple animal models, including dizocilpine (MK-801) induced hyper-locomotion; APO induced climbing; Conditioned Avoidance Response (CAR); DOI-Induced Head Twitch; Forced swimming test; Scopolamine induced cognitive impairment model, were used to verify the antipsychotic activity of NH300094 in preclinical. Results: In vitro functional assays have indicated that NH300094 is a potent antagonist of 5-HT receptors and dopamine receptors, with higher relative antagonistic activity against 5-HT2A receptor (5-HT2A IC50 = 0.47 nM) than dopamine receptors (D2L IC50 = 1.04 nM; D2S IC50 = 11.71 nM; D3 IC50 = 31.55 nM). Preclinical in vivo pharmacological study results showed that NH300094 was effective in multiple models, which is more extensive than the clinic drug Risperidone. Furthermore, the safety window for extrapyramidal side effects of NH300094 is significantly wider than that of Risperidone (For NH300094, mice catalepsy model ED50/ Mice MK-801 model ED50 = 104.6-fold; for Risperidone, mice catalepsy model ED50/ Mice MK-801 model ED50 = 12.9-fold), which suggests a potentially better clinical safety profile for NH300094. Conclusion: NH300094 is a novel potent serotonin and dopamine receptors modulator, which has good safety profile and therapeutic potential for the treatment of schizophrenia with cognition disorders.

Phase-Change Based Oxygen Carriers Improve Acute Cerebral Hypoxia.

Stroke is the second leading cause of death worldwide, and hypoxia is a major crisis of the brain after stroke. Therefore, providing oxygen to the brain microenvironment can effectively protect neurons from damage caused by cerebral hypoxia. However, there is a lack of timely and effective means of oxygen delivery clinically to the brain for acute cerebral hypoxia. Here, a phase-change based nano oxygen carrier is reported, which can undergo a phase change in response to increasing temperature in the brain, leading to oxygen release. The nano oxygen carrier demonstrate intracerebral oxygen delivery capacity and is able to release oxygen in the hypoxic and inflammatory region of the brain. In the acute ischemic stroke mouse model, the nano oxygen carrier can effectively reduce the area of cerebral infarction and decrease the level of inflammation triggered by cerebral hypoxia. By taking advantage of the increase in temperature during cerebral hypoxia, phase-change oxygen carrier proposes a new intracerebral oxygen delivery strategy for reducing acute cerebral hypoxia.

Antigen-loaded flagellate bacteria for enhanced adaptive immune response by intradermal injection.

Since the skin limits the distribution of intradermal vaccines, a large number of dendritic cells in the skin cannot be fully utilized to elicit a more effective immune response. Here, we loaded the antigen to the surface of the flagellate bacteria that was modified by cationic polymer, thus creating antigen-loaded flagellate bacteria (denoted as 'FB-Ag') to overcome the skin barrier and perform the active delivery of antigen in the skin. The FB-Ag showed fast speed (∼0.2 µm s-1) and strong dendritic cell activation capabilities in the skin model in vitro. In vivo, the FB-Ag promoted the spread of antigen in the skin through active movement, increased the contact between Intradermal dendritic cells and antigen, and effectively activated the internal dendritic cells in the skin. In a mouse of pulmonary metastatic melanoma and in mice bearing subcutaneous melanoma tumor, the FB-Ag effectively increased antigen-specific therapeutic efficacy and produced long-lasting immune memory. More importantly, the FB-Ag also enhanced the level of COVID-19 specific antibodies in the serum and the number of memory B cells in the spleen of mice. The movement of antigen-loaded flagellate bacteria to overcome intradermal constraints may enhance the activation of intradermal dendritic cells, providing new ideas for developing intradermal vaccines.

NLRP6 potentiates PI3K/AKT signalling by promoting autophagic degradation of p85α to drive tumorigenesis.

The PI3K/AKT pathway plays an essential role in tumour development. NOD-like receptors (NLRs) regulate innate immunity and are implicated in cancer, but whether they are involved in PI3K/AKT pathway regulation is poorly understood. Here, we report that NLRP6 potentiates the PI3K/AKT pathway by binding and destabilizing p85α, the regulatory subunit of PI3K. Mechanistically, NLRP6 recruits the E3 ligase RBX1 to p85α and ubiquitinates lysine 256 on p85α, which is recognized by the autophagy cargo receptor OPTN, causing selective autophagic degradation of p85α and subsequent activation of the PI3K/AKT pathway by reducing PTEN stability. We further show that loss of NLRP6 suppresses cell proliferation, colony formation, cell migration, and tumour growth in glioblastoma cells in vitro and in vivo. Disruption of the NLRP6/p85α interaction using the Pep9 peptide inhibits the PI3K/AKT pathway and generates potent antitumour effects. Collectively, our results suggest that NLRP6 promotes p85α degradation via selective autophagy to drive tumorigenesis, and the interaction between NLRP6 and p85α can be a promising therapeutic target for tumour treatment.

Long-term and liver-selected ginsenoside C-K nanoparticles retard NAFLD progression by restoring lipid homeostasis.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent hepatic disease characterized as lipid accumulation, yet without any approved drug. And development of therapeutic molecules is obstructed by low efficiency and organ toxicity. Herein, we develop a long-term, low-toxic and liver-selected nano candidate, nabCK, to alleviate NAFLD. NabCK is simply composed by natural compound ginsenoside compound K (CK) and albumin. As a major metabolite of ginseng, ginsenoside CK has excellently modulating functions for lipid metabolism, but accompanied by an extremely poor bioavailability <1%. Albumin is a key lipid carrier secreted and metabolized by livers. Thereby, it can improve solubility and liver-localization of CK. In adipocytes and hepatocytes, nabCK prevents lipid deposition and eliminates lipid droplets. Transcriptomic analysis reveals that nabCK rectifies various pathways that involved in steatosis development, including lipid absorption, lipid export, fatty acid biosynthesis, lipid storage and inflammation. All these pathways are modulated by mTOR, the pivotal feedback sensor that is hyperactive in NAFLD. NabCK suppresses mTOR activation to restores lipid homeostasis. In high-fat diet (HFD) induced NAFLD mice, nabCK retards development of steatosis and fibrosis, coupling a protective effect on cardiac tissues from lipotoxicity. Together, nabCK is a safe and potent candidate to offer benefits for NAFLD treatment.

Flagella of Tumor-Targeting Bacteria Trigger Local Hemorrhage to Reprogram Tumor-Associated Macrophages for Improved Antitumor Therapy.

Tumor-associated macrophages (TAMs) exhibit an immunosuppressive M2 phenotype and lead to failure of antitumor therapy. Infiltrated erythrocytes during hemorrhage are recognized as a promising strategy for polarizing TAMs. However, novel materials that precisely induce tumor hemorrhage without affecting normal coagulation still face challenges. Here, tumor-targeting bacteria (flhDC VNP) are genetically constructed to realize precise tumor hemorrhage. FlhDC VNP colonizes the tumor and overexpresses flagella during proliferation. The flagella promote the expression of tumor necrosis factor α, which induces local tumor hemorrhage. Infiltrated erythrocytes during the hemorrhage temporarily polarize macrophages to the M1 subtype. In the presence of artesunate, this short-lived polarization is transformed into a sustained polarization because artesunate and heme form a complex that continuously produces reactive oxygen species. Therefore, the flagella of active tumor-targeting bacteria may open up new strategies for reprogramming TAMs and improving antitumor therapy.

Combining High-Z Sensitized Radiotherapy with CD73 Blockade to Boost Tumor Immunotherapy.

Radiation therapy (RT) has the capacity to induce immunogenic death in tumor cells, thereby potentially inducing in situ vaccination (ISV) to prime systemic antitumor immune responses. However, RT alone is often faced with various limitations during ISV induction, such as insufficient X-ray deposition and an immunosuppressive microenvironment. To overcome these limitations, we constructed nanoscale coordination particles AmGd-NPs by self-assembling high-Z metal gadolinium (Gd) and small molecular CD73 inhibitor AmPCP. Then, AmGd-NPs could synergize with RT to enhance immunogenic cell death, improve phagocytosis, and promote antigen presentation. Additionally, AmGd-NPs could also gradually release AmPCP to inhibit CD73's enzymatic activity and prevent the conversion of extracellular ATP to adenosine (Ado), thereby driving a proinflammatory tumor microenvironment that promotes DC maturation. As a result, AmGd-NPs sensitized RT induced potent in situ vaccination and boosted CD8+ T cell-dependent antitumor immune responses against both primary and metastatic tumors, which could also be potentiated by immune checkpoint inhibitory therapy.

In Situ Vaccination with Mitochondria-Targeting Immunogenic Death Inducer Elicits CD8+ T Cell-Dependent Antitumor Immunity to Boost Tumor Immunotherapy.

In situ vaccination can elicit systemic antitumor immunity to potentiate immune checkpoint blockade (ICB) in poorly immunogenic tumors. Herein, an immunogenic cell death (ICD) inducer for in situ vaccination, which is based on a mitochondria-targeting modification of fenofibric acid (FFa), a lipid-lowering drug with potential inhibitory efficacy of respiratory complex I is developed. Mitochondria-targeting FFa (Mito-FFa) inhibits complex I efficiently and increases mitochondrial ROS (mtROS) generation, which further triggers endoplasmic reticulum (ER) stress with unprecedented calreticulin (CRT) exposure on tumor cellular membranes. Moreover, the generated mtROS also oxidizes mitochondrial DNA (mtDNA) and promotes it leakage into the cytoplasm for cGAS-STING-dependent type I interferon (IFN-I) secretion. The synchronous CRT exposure and IFN-I secretion successively improve the uptake of tumor antigens, maturation of dendritic cells (DCs) and cross-priming of CD8+ T cells. In a poorly immunogenic 4T1 tumor model, a single intratumoral (i.t.) Mito-FFa injection turns immune-cold tumors into hot ones and elicits systemic tumor-specific CD8+ T cells responses against primary and metastatic tumors. Furthermore, the synergistic effect with PD-L1 blockade and good bio-safety of i.t. Mito-FFa administration suggest the great translational potential of Mito-FFa in tumor immunotherapy.

Exploration of NPC2 as a Potential Biomarker for Immunotherapy Using RNA-seq and Protein Data - A New Hypothesis.

INTRODUCTION: NPC2 is well known as a player for cholesterol transport. However, the biological role of NPC2 in cancer development and therapy is far from clear. METHODS: Here, we explore the potential role of NPC2 in prognosis and immunotherapy across multiple cancer types by integrating RNA-seq data from TCGA and GTEx, protein data from CPTAC, and multiple web analysis databases. RESULTS: Expression depiction between tumour and normal tissues indicated that NPC2 is overexpressed in the majority of the most common cancer types, including glioblastoma and pancreatic cancer, two cancers mostly difficult to diagnose and treat. CONCLUSION: Cancer stemness in glioblastoma is negatively associated with NPC2 level. NPC2 expression is positively correlated with immune cell infiltration and the expression of several immune checkpoints. IDH1 mutation in GBM is negatively correlated with NPC2 level, while a positive correlation has been found between TP53 mutation and NPC2 expression in pancreatic cancer. NPC2 is also correlated with levels of serum biomarkers used for diagnosis of pancreatic cancer.

Endovascular treatment achieves better outcomes than best medical management in patients with M2 occlusion and high stroke severity: a meta-analysis.

BACKGROUND: The lack of randomized evidence makes it difficult to establish reliable treatment recommendations for patients with M2 occlusion. This study aims to compare the efficacy and safety of endovascular treatment (EVT) with best medical management (BMM) in patients with M2 occlusion, and to investigate whether the optimal treatment varies according to stroke severity. METHODS: Comprehensive literature retrieval was conducted to identify studies that directly compared the outcomes of EVT and BMM. According to stroke severity, the study population were classified into those with moderate-severe stroke and those with mild stroke. National Institute of Health Stroke Scale (NIHSS) scores ≥ 6 was defined as moderate-severe stroke, and NIHSS scores 0-5 as mild stroke. Random-effects meta-analyses were performed to measure the symptomatic intracranial hemorrhage (sICH) within 72 h, and the modified Rankin Scale (mRS) scores 0-2 and the mortality at 90 days. RESULTS: Totally, 20 studies were identified, including 4358 patients. In the moderate-severe stroke population, the EVT had 82% higher odds for mRS scores 0-2 (OR 1.82, 95% CI 1.34-2.49) and a 43% lower odds for mortality (OR 0.57, 95% CI 0.39-0.82) compared with the BMM. However, no difference was found in the sICH rate (OR 0.88, 95% CI 0.44-1.77). In the mild stroke population, no differences were observed in the mRS scores 0-2 (OR 0.81, 95% CI 0.59-1.10) or mortality (OR 1.23, 95% CI 0.72-2.10) between EVT and BMM, whereas EVT was associated with higher sICH rate (OR 4.21, 95% CI 1.86-9.49). CONCLUSION: EVT may be only beneficial for patients with M2 occlusion and high stroke severity, but not for those with NIHSS scores 0-5.

Case report: Identification of novel fibrillin-2 variants impacting disulfide bond and causing congenital contractural arachnodactyly.

Background: Congenital contractural arachnodactyly (CCA) is an autosomal dominant connective tissue disorder with clinical features of arthrogryposis, arachnodactyly, crumpled ears, scoliosis, and muscular hypoplasia. The heterozygous pathogenic variants in FBN2 have been shown to cause CCA. Fibrillin-2 is related to the elasticity of the tissue and has been demonstrated to play an important role in the constitution of extracellular microfibrils in elastic fibers, providing strength and flexibility to the connective tissue that sustains the body's joints and organs. Methods: We recruited two Chinese families with arachnodactyly and bilateral arthrogryposis of the fingers. Whole-exome sequencing (WES) and co-segregation analysis were employed to identify their genetic etiologies. Three-dimensional protein models were used to analyze the pathogenic mechanism of the identified variants. Results: We have reported two CCA families and identified two novel missense variants in FBN2 (NM_001999.3: c.4093T>C, p.C1365R and c.2384G>T, p.C795F). The structural models of the mutant FBN2 protein in rats exhibited that both the variants could break disulfide bonds. Conclusion: We detected two FBN2 variants in two families with CCA. Our description expands the genetic profile of CCA and emphasizes the pathogenicity of disulfide bond disruption in FBN2.

Cytosolic perfluorocarbon delivery to platelets via albumin for antithrombotic therapy.

Thrombosis is a major contributor to global disease burden. Antiplatelet therapy is the critical approach to prevent thrombosis by reducing platelet reactivity. However, classical antiplatelet strategies generally interfere with platelet integrin αIIbß3-mediated platelet activation, thereby facing severe bleeding risk. To break the limitation, we described an integrin αIIbß3-independent antiplatelet method by cytosolic delivery of nanoscale perfluorocarbon (PFC) to platelets via albumin carrier. Denatured albumin was found to build high affinity with platelets to mediate cytosolic PFC delivery. While, cytosolic PFC impaired cytoskeleton reorganization during platelet activation to inhibit relevant platelet functions, but avoided to interfere with integrin αIIbß3. We proved that this αIIbß3-indenpendent antiplatelet pattern showed potential antiplatelet effect with low bleeding risk to prevent thrombosis in various thrombosis models. Together, cytosolic PFC delivery via albumin is a promising antiplatelet approach, and will provide an alternative regimen for current antithrombotic therapy.

An In Situ Assembled Trapping Gel Repairs Spinal Cord Injury by Capturing Glutamate and Free Calcium Ions.

Spinal cord injury (SCI) can lead to devastating autonomic dysfunction. One of the most challenging issues for functional repair in SCI is the secondary damage caused by the increased release of glutamate and free Ca2+ from injured cells. Here, an in situ assembled trapping gel (PF-SA-GAD) is developed to sweep glutamate and Ca2+ , promoting SCI repair. The hydrogel solution is a mixture of recombinant glutamate decarboxylase 67 (rGAD67) protein, sodium alginate (SA), and pluronic F-127 (PF-127). After intrathecal administration, temperature-sensitive PF-127 promoted in situ gelation. Glutamate (Glu) is captured and decarboxylated by rGAD67 into γ-aminobutyric acid (GABA). SA reacted with the free Ca2+ to generate gellable calcium alginate. Thereby, this in situ trapping gel retarded secondary neuron injury caused by Glu and free Ca2+ during SCI. In rat models of SCI, PF-SA-GAD reduces the lesion volume and inflammatory response after SCI, restores the motor function of rats with SCI. Together, the in situ assembled trapping gel is a long-term effective and minimally invasive sweeper for the direct elimination of glutamate and Ca2+ from injury lesions and can be a novel strategy for SCI repair by preventing secondary injury.

Hijacking Self-Assembly to Establish Intracellular Functional Nanoparticles.

The targeted transport of nanomedicines is often impeded by various biological events in the body. Viruses can hijack host cells and utilize intracellular transcription and translation biological events to achieve their replication. Inspired by this, a strategy to hijack endogenous products of biological events to assemble into intracellular functional nanoparticles is established. It has been shown that, following tumor vessel destruction therapy, injected cell permeable small molecule drugs bisphosphonate can hijack the hemorrhagic product iron and self-assemble into peroxidase-like nanoparticles within tumor-infiltrating macrophages. Unlike free drugs, the generated intercellular nanoparticles can specifically stress mitochondria, resulting in immune activation of macrophages in vitro and polarizing tumor-associated macrophages (TAMs) from immunosuppressive to tumoricidal and increasing the recruitment of T cells deep within tumor. The hijacking self-assembly strategy significantly inhibits tumor growth compared with the treatment of vascular-disrupting agents alone. Using bisphosphonate to hijack the metabolite associated with hemorrhage, iron, to fabricate functional nanoparticles within specific cells, which may open up new nanotechnology for drug delivery and small molecular drug development.

Novel heterozygous mutation in COL4A4 responsible for Alport syndrome in a Chinese family.

Background: Chronic kidney disease, a global public health problem, results in kidney damage or a gradual decline in the glomerular filtration rate. Alport syndrome is commonly characterized by chronic glomerulonephritis caused by a structural disorder in the glomerular basement membrane. Currently, three disease-causing genes, namely collagen type IV alpha 3-5 (COL4A3, COL4A4, and COL4A5), have been associated with the occurrence of Alport syndrome. Methods: We enrolled a Chinese family where the affected individuals suffered from recurrent hematuria and proteinuria. The proband was selected for whole-exome sequencing to identify the pathogenic mutations in this family. Results: After data filtering, a novel heterozygous COL4A4 variant (NM_000092: c.853G>A/p. G285A) was identified as the putative genetic lesion in the affected individuals. Further co-segregation analysis using Sanger sequencing confirmed that this novel COL4A4 mutation (c.853G>A/p. G285A) exists only in the affected individuals and is absent in other healthy family members as well as in the control cohort of 200 individuals from the same locality. According to American College of Medical Genetics and Genomics guidelines, the mutation was classified as 'potentially pathogenic'. A bioinformatics-based prediction analysis revealed that this mutation is pathogenic and may disrupt the structure and function of type IV collagen. This variant is located at an evolutionarily conserved site of COL4A4. Conclusion: In this study, we identified a novel heterozygous COL4A4 variant (c.853G>A) in a Chinese AS family and assisted to diagnose this AS proband as autosomal-dominant Alport syndrome (ADAS). Our study expands the spectrum of Alport syndrome mutations and contributes to the genetic counseling and diagnosis of patients with Alport syndrome.

Flagellated bacterial porter for in situ tumor vaccine.

Cancer immunotherapy, which use the own immune system to attack tumors, are increasingly popular treatments. But, due to the tumor immunosuppressive microenvironment, the antigen presentation in the tumor is limited. Recently, a growing number of people use bacteria to stimulate the body's immunity for tumor treatment due to bacteria themselves have a variety of elements that activate Toll-like receptors. Here, we discuss the use of motility of flagellate bacteria to transport antigens to the tumor periphery to activate peritumoral dendritic cells to enhance the effect of in situ tumor vaccines.

Iron-based nanoscale coordination polymers synergistically induce immunogenic ferroptosis by blocking dihydrofolate reductase for cancer immunotherapy.

Iron is indispensable for cancer cell survival and cancer cells are more vulnerable to ferroptosis than normal cells. Ferroptosis holds promise for overcoming chemoresistance and inducing tumor immunogenic cell death, which offers new possibilities for cancer immunotherapy. However, the prevalence of immunogenic ferroptosis in cancer cells is diminished because of the high levels of reducing substances within tumor microenvironments. Ferroptosis-needed iron is overdose for livings, which is also an obstacle for effective immune responses. In this study, we construct self-assembled carrier-free nanoscale coordination polymers based on iron and methotrexate (MFe-NCPs). The low-dose-iron-induced immunogenic ferroptosis is obviously enhanced by methotrexate via inhibiting dihydrofolate reductase and abating substance reduction, respectively. Of note, MFe-NCPs sequentially promoted antigen presentation, immune activation, T cell infiltration and boosted the therapeutic effect of immune checkpoint blockade therapy.