Enlightenment of COVID-19 Treated by Botanical Drugs on the Development of Drugs for Rare Diseases in China

Objective To study the feasibility of developing botanical drugs to treat intractable diseases and play an important role in dealing with major public health crises.Methods From January 1990 to May 2021,a bibliographic search was carried out on the use of botanical drugs,rare disease drugs,related registration management policies and regulations in PubMed and CNKI.The following keywords were searched in the database:Rare disease policies and regulations,orphan drugs,botanical drugs for intractable diseases,botanical drugs for the treatment of new coronary pneumonia,traditional Chinese medicine,and emergency guidelines for major public health crisis.Other data were obtained from“Chinese Pharmacopoeia”and relevant Chinese government websites for sorting and analysis.Results and Conclusion Based on 39 Chinese corresponding policies and regulations,challenges and opportunities of developing and researching drugs for treating rare diseases were found out after the analysis and comparison.Based on the study of national policies on drugs for rare diseases,the priority review and approval procedures in the drug registration,as well as China’s emergency guidelines and policies for major public health events,some problems in the use of drugs for rare diseases are found out.Therefore,it is recommended to actively adopt the property rights protection system,explore the folk prescriptions of traditional Chinese medicine and the potential of hospital preparations,and the registration review strategy of giving priority to the use of botanical drugs for rare diseases.Thus,the international status of botanical drugs for rare disease and the influence of responding to major public health events can be enhanced.

Strategies for translating proteomics discoveries into drug discovery for dementia

Tauopathies,diseases characterized by neuropathological aggregates of tau including Alzheimer's disease and subtypes of fro ntotemporal dementia,make up the vast majority of dementia cases.Although there have been recent developments in tauopathy biomarkers and disease-modifying treatments,ongoing progress is required to ensure these are effective,economical,and accessible for the globally ageing population.As such,continued identification of new potential drug targets and biomarkers is critical."Big data"studies,such as proteomics,can generate information on thousands of possible new targets for dementia diagnostics and therapeutics,but currently remain underutilized due to the lack of a clear process by which targets are selected for future drug development.In this review,we discuss current tauopathy biomarkers and therapeutics,and highlight areas in need of improvement,particularly when addressing the needs of frail,comorbid and cognitively impaired populations.We highlight biomarkers which have been developed from proteomic data,and outline possible future directions in this field.We propose new criteria by which potential targets in proteomics studies can be objectively ranked as favorable for drug development,and demonstrate its application to our group's recent tau interactome dataset as an example.

Biomaterials-based anti-inflammatory treatment strategies for Alzheimer's disease

The current therapeutic drugs for Alzheimer's disease only improve symptoms,they do not delay disease progression.Therefo re,there is an urgent need for new effective drugs.The underlying pathogenic factors of Alzheimer's disease are not clear,but neuroinflammation can link various hypotheses of Alzheimer's disease;hence,targeting neuroinflammation may be a new hope for Alzheimer's disease treatment.Inhibiting inflammation can restore neuronal function,promote neuro regeneration,reduce the pathological burden of Alzheimer's disease,and improve or even reverse symptoms of Alzheimer's disease.This review focuses on the relationship between inflammation and various pathological hypotheses of Alzheimer's disease;reports the mechanisms and characteristics of small-molecule drugs(e.g.,nonsteroidal anti-inflammatory drugs,neurosteroids,and plant extracts);macromolecule drugs(e.g.,peptides,proteins,and gene therapeutics);and nanocarriers(e.g.,lipid-based nanoparticles,polymeric nanoparticles,nanoemulsions,and inorganic nanoparticles)in the treatment of Alzheimer's disease.The review also makes recommendations for the prospective development of anti-inflammatory strategies based on nanocarriers for the treatment of Alzheimer's disease.

Neg-Entropy Mechanism as a Target for Natural Medicines

Current strategies for drug discovery research mainly focus on molecular targets,in which structure biology plays a central role,such as the detailed mechanism of a drug-target interaction.The principle of this approach is the presumed molecular mechanism or genetic background of the disease being treated.Although this approach has brought progress in understanding the mode of action for some drugs,most prevalent illnesses-especially chronic diseases-are multifactorial and present with a group of symptoms,as is often the case in cardiovascular diseases,metabolic disorders,cancers,and so forth.The newly emerging term“multi-morbidity”(i.e.,multiple co-occurring diseases)describes a grand challenge for this strategy,and successful drug intervention for multimorbidity should be multifaceted[1].So,our research team has shifted its attention to a new direction that views the disease as a whole instead of a single-molecule abnormality,and focusing on drugs with multiple targets.

Liposomes as versatile agents for the management of traumatic and nontraumatic central nervous system disorders:drug stability,targeting efficiency,and safety

Various nanoparticle-based drug delivery systems for the treatment of neurological disorders have been widely studied.However,their inability to cross the blood–brain barrier hampers the clinical translation of these therapeutic strategies.Liposomes are nanoparticles composed of lipid bilayers,which can effectively encapsulate drugs and improve drug delivery across the blood–brain barrier and into brain tissue through their targeting and permeability.Therefore,they can potentially treat traumatic and nontraumatic central nervous system diseases.In this review,we outlined the common properties and preparation methods of liposomes,including thin-film hydration,reverse-phase evaporation,solvent injection techniques,detergent removal methods,and microfluidics techniques.Afterwards,we comprehensively discussed the current applications of liposomes in central nervous system diseases,such as Alzheimer's disease,Parkinson's disease,Huntington's disease,amyotrophic lateral sclerosis,traumatic brain injury,spinal cord injury,and brain tumors.Most studies related to liposomes are still in the laboratory stage and have not yet entered clinical trials.Additionally,their application as drug delivery systems in clinical practice faces challenges such as drug stability,targeting efficiency,and safety.Therefore,we proposed development strategies related to liposomes to further promote their development in neurological disease research.

Controlled release of dexamethasone from fibrin sealant for intratympanic administration in inner ear therapy

The aim of the present work was to show the sustainability of fibrin sealant in releasing dexamethasone and adjust the protocol for clinical application of the novel method in the treatment of Meniere’s disease (MD) and sudden sensorineural hearing loss (SSHL).Gelation occurred shortly after mixing dexamethasone-containing fibrinogen with thrombin.Dexamethasone was constantly released for at least 16 d at a stable level after 7d in protocol 1 (low-dose),while it was robustly released within 4 d and slowed afterward until 10 d in protocol 2(high-dose).There were significant differences among the time points in Protocol 2 (p<0.01,ANOVA),and the exponential model with the formula y=15.299*e~(-0.483*t) fits the association.The estimated concentration of dexamethasone released on 7 d in protocol 2 was slightly lower than that observed in protocol 1.The fibrin sealant is capable of constantly releasing dexamethasone with adjustable dynamics.Targeted and minimally invasive administration of the material can be achieved in the clinic by sequential injections of the fluids using a soft-tipped catheter.

Emerging structures and dynamic mechanisms ofγ-secretase for Alzheimer’s disease

γ-Secretase,called“the proteasome of the membrane,”is a membrane-embedded protease complex that cleaves 150+peptide substrates with central roles in biology and medicine,including amyloid precursor protein and the Notch family of cell-surface receptors.Mutations inγ-secretase and amyloid precursor protein lead to early-onset familial Alzheimer’s disease.γ-Secretase has thus served as a critical drug target for treating familial Alzheimer’s disease and the more common late-onset Alzheimer’s disease as well.However,critical gaps remain in understanding the mechanisms of processive proteolysis of substrates,the effects of familial Alzheimer’s disease mutations,and allosteric modulation of substrate cleavage byγ-secretase.In this review,we focus on recent studies of structural dynamic mechanisms ofγ-secretase.Different mechanisms,including the“Fit-Stay-Trim,”“Sliding-Unwinding,”and“Tilting-Unwinding,”have been proposed for substrate proteolysis of amyloid precursor protein byγ-secretase based on all-atom molecular dynamics simulations.While an incorrect registry of the Notch1 substrate was identified in the cryo-electron microscopy structure of Notch1-boundγ-secretase,molecular dynamics simulations on a resolved model of Notch1-boundγ-secretase that was reconstructed using the amyloid precursor protein-boundγ-secretase as a template successfully capturedγ-secretase activation for proper cleavages of both wildtype and mutant Notch,being consistent with biochemical experimental findings.The approach could be potentially applied to decipher the processing mechanisms of various substrates byγ-secretase.In addition,controversy over the effects of familial Alzheimer’s disease mutations,particularly the issue of whether they stabilize or destabilizeγ-secretase-substrate complexes,is discussed.Finally,an outlook is provided for future studies ofγ-secretase,including pathways of substrate binding and product release,effects of modulators on familial Alzheimer’s disease mutations of theγ-secretase-substrate complexes.Comprehensive understanding of the functional mechanisms ofγ-secretase will greatly facilitate the rational design of effective drug molecules for treating familial Alzheimer’s disease and perhaps Alzheimer’s disease in general.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Nanoparticles for the treatment of spinal cord injury

Spinal cord injuries lead to significant loss of motor, sensory, and autonomic functions, presenting major challenges in neural regeneration. Achieving effective therapeutic concentrations at injury sites has been a slow process, partly due to the difficulty of delivering drugs effectively. Nanoparticles, with their targeted delivery capabilities, biocompatibility, and enhanced bioavailability over conventional drugs, are garnering attention for spinal cord injury treatment. This review explores the current mechanisms and shortcomings of existing treatments, highlighting the benefits and progress of nanoparticle-based approaches. We detail nanoparticle delivery methods for spinal cord injury, including local and intravenous injections, oral delivery, and biomaterial-assisted implantation, alongside strategies such as drug loading and surface modification. The discussion extends to how nanoparticles aid in reducing oxidative stress, dampening inflammation, fostering neural regeneration, and promoting angiogenesis. We summarize the use of various types of nanoparticles for treating spinal cord injuries, including metallic, polymeric, protein-based, inorganic non-metallic, and lipid nanoparticles. We also discuss the challenges faced, such as biosafety, effectiveness in humans, precise dosage control, standardization of production and characterization, immune responses, and targeted delivery in vivo. Additionally, we explore future directions, such as improving biosafety, standardizing manufacturing and characterization processes, and advancing human trials. Nanoparticles have shown considerable progress in targeted delivery and enhancing treatment efficacy for spinal cord injuries, presenting significant potential for clinical use and drug development.

The Oral Health Status of Person with Special Needs in Kumasi

Background: The most prevalent non-communicable disease worldwide is oral health-related disease, with dental caries and periodontal conditions being common. Oral health status significantly impacts overall health and quality of life. Barriers to dental care affect children with special needs. This study evaluates the oral health status and treatment requirements of people with special needs. Methodology: This descriptive cross-sectional study in Kumasi, Ghana examined oral health status among people, 121 children with special needs over two months, in June-July 2022. Dental caries, periodontal changes, malocclusion, and trauma were assessed. The data was analyzed using SPSS 20.0. Results: The oral health status among people with special needs in this study was generally moderate, with a range from very good to very poor. The prevalence of dental caries was 37%, with a mean DMFT score of 2.82. Periodontal health showed unsatisfactory indicators such as mobile teeth, poor gingival health, high plaque scores, and the presence of halitosis. Conclusion: This descriptive study aimed to provide baseline data on the oral health status of special needs students in Kumasi, addressing the lack of knowledge in this area. It highlighted the importance of dental care in maintaining good oral health and overall well-being in individuals with special needs. Coordinated efforts by dental professionals are needed to provide dental health education and preventive interventions for these children.

Is tumor necrosis factor-α monoclonal therapy with proactive therapeutic drug monitoring optimized for inflammatory bowel disease? Network meta-analysis

BACKGROUND The efficacy and safety of anti-tumor necrosis factor-α(TNF-α)monoclonal antibody therapy[adalimumab(ADA)and infliximab(IFX)]with therapeutic drug monitoring(TDM),which has been proposed for inflammatory bowel disease(IBD)patients,are still controversial.AIM To determine the efficacy and safety of anti-TNF-αmonoclonal antibody therapy with proactive TDM in patients with IBD and to determine which subtype of IBD patients is most suitable for proactive TDM interventions.METHODS As of July 2023,we searched for randomized controlled trials(RCTs)and observa-tional studies in PubMed,Embase,and the Cochrane Library to compare anti-TNF-αmonoclonal antibody therapy with proactive TDM with therapy with reactive TDM or empiric therapy.Pairwise and network meta-analyses were used to determine the IBD patient subtype that achieved clinical remission and to determine the need for surgery.RESULTS This systematic review and meta-analysis yielded 13 studies after exclusion,and the baseline indicators were balanced.We found a significant increase in the number of patients who achieved clinical remission in the ADA[odds ratio(OR)=1.416,95%confidence interval(CI):1.196-1.676]and RCT(OR=1.393,95%CI:1.182-1.641)subgroups and a significant decrease in the number of patients who needed surgery in the proactive vs reactive(OR=0.237,95%CI:0.101-0.558)and IFX+ADA(OR=0.137,95%CI:0.032-0.588)subgroups,and the overall risk of adverse events was reduced(OR=0.579,95%CI:0.391-0.858)according to the pairwise meta-analysis.Moreover,the network meta-analysis results suggested that patients with IBD treated with ADA(OR=1.39,95%CI:1.19-1.63)were more likely to undergo TDM,especially in comparison with patients with reactive TDM(OR=1.38,95%CI:1.07-1.77).CONCLUSION Proactive TDM is more suitable for IBD patients treated with ADA and has obvious advantages over reactive TDM.We recommend proactive TDM in IBD patients who are treated with ADA.

Call for papers of the special issue on Animal Models and Infectious Diseases

Primates and animal models are major areas of coverage for Zoological Research （ZR）. Over the past few years, ZR has released a series of special issues/topics addressing various aspects of these areas, e.g., ge- netics, immunology, and physiology neuroscience. A special issue for 2017 focusing on ＂Animal Models of Infectious Diseases＂ is under preparation and, so far, includes original research articles and reviews on filo- viruses and coxsackievirus involving guinea pigs, mice, and other species. Further to this, ZR would like to extend a very warm invitation to all peer researchers in the field to submit outstanding work to the journal on this special issue.

Circ RNA and ferroptosis in human disease:Insights for new treatments

Circular RNA(circ RNA),classified as a type of non-coding RNA,has gained significant attention in the field of biology due to its distinctive ring structure and functional properties.Recent research has provided evidence that specific circ RNAs have the ability to modulate disease progression through diverse mechanisms,one of which is by regulating cellular ferroptosis.Ferroptosis is a form of regulated cell death that is driven by iron dependency and lipid peroxidation,and extensive investigations have revealed a relationship between ferroptosis and disease development.In addition to evidence that both circ RNAs and ferroptosis exert critical roles in disease progression,circ RNAs have also been shown to actively mediate the process of ferroptosis.The relationship between circ RNAs and ferroptosis therefore influences disease progression and offers novel targets for disease treatment.By directly or indirectly modulating the expression of circ RNAs that regulate the expression of ferroptosisrelated proteins,it may be possible to impact disease progression by promoting or inhibiting ferroptosis.Current research indicates such approaches may hold significant value in a wide variety of common diseases across physiological systems.This review comprehensively summarizes the findings of recent studies investigating the roles of circ RNAs in the regulation of ferroptosis in various diseases.

Potential therapeutic targets for nonalcoholic fatty liver disease:Glucagon-like peptide 1

Nonalcoholic fatty liver disease(NAFLD)is the most rapidly growing contributor to liver mortality and morbidity.Hepatocellular injury in nonalcoholic steatohepatitis(NASH)is caused by an increase in metabolic substrates(glucose,fructose,and fatty acids),leading fatty acids to participate in pathways that cause cellular injury and a poor response to injury.The pathogenesis of this disease is largely associated with obesity,type 2 diabetes,and increasing age.To date,there are no Food and Drug Administration-approved treatments for NAFLD/NASH or its associated fibrosis.Since one of the pathogenic drivers of NASH is insulin resistance,therapies approved for the treatment of type 2 diabetes are being evaluated in patients with NASH.Currently,the glucagon-like peptide-1 receptor agonist(GLP-1RA)semaglutide is a safe,well-studied therapeutic for NAFLD/NASH patients.Existing research demonstrates that semaglutide can increase the resolution of NASH but not improve fibrosis.However,improving the fibrosis of NAFLD is the only way to improve the long-term prognosis of NAFLD.Given the complex pathophysiology of NASH,combining therapies with complementary mechanisms may be beneficial.Researchers have conducted trials of semaglutide in combination with antifibrotic drugs.However,the results have not fully met expectations,and it cannot be ruled out that the reason is the short trial time.We should continue to pay increasing attention to GLP-1RAs.

Repositioning of clinically approved drug Bazi Bushen capsule for treatment of Alzheimer′s disease using network pharma⁃cology approach and in vitro experimental validation

OBJECTIVE To explore the new indications and key mechanism of Bazi Bushen capsule(BZBS)by network pharmacology and in vitro experiment.METHODS The potential tar⁃get profiles of the components of BZBS were pre⁃dicted.Subsequently,new indications for BZBS were predicted by disease ontology(DO)enrich⁃ment analysis and initially validated by GO and KEGG pathway enrichment analysis.Further⁃more,the therapeutic target of BZBS acting on AD signaling pathway were identified by intersec⁃tion analysis.Two Alzheimer′s disease(AD)cell models,BV-2 and SH-SY5Y,were used to pre⁃liminarily verify the anti-AD efficacy and mecha⁃nism of BZBS in vitro.RESULTS In total,1499 non-repeated ingredients were obtained from 16 herbs in BZBS formula,and 1320 BZBS targets with high confidence were predicted.Disease enrichment results strongly suggested that BZBS formula has the potential to be used in the treat⁃ment of AD.In vitro experiments showed that BZ⁃BS could significantly reduce the release of TNF-αand IL-6 and the expression of COX-2 and PSEN1 in Aβ25-35-induced BV-2 cells.BZBS reduced the apoptosis rate of Aβ25-35 induced SH-SY5Y cells,significantly increased mitochon⁃drial membrane potential,reduced the expres⁃sion of Caspase3 active fragment and PSEN1,and increased the expression of IDE.CONCLU⁃SIONS BZBS formula has a potential use in the treatment of AD,which is achieved through regu⁃lation of ERK1/2,NF-κB signaling pathways,and GSK-3β/β-catenin signaling pathway.Further⁃more,the network pharmacology technology is a feasible drug repurposing strategy to reposition new clinical use of approved TCM and explore the mechanism of action.The study lays a foun⁃dation for the subsequent in-depth study of BZBS in the treatment of AD and provides a basis for its application in the clinical treatment of AD.

Death-associated protein kinase 1 is associated with cognitive dysfunction in major depressive disorder

We previously showed that death-associated protein kinase 1(DAPK1)expression is increased in hippocampal tissue in a mouse model of major depressive disorde and is related to cognitive dysfunction in Alzheimer's disease.In addition,depression is a risk factor for developing Alzheimer's disease,as well as an early clinical manifestation of Alzheimer's disease.Meanwhile,cognitive dysfunction is a distinctive feature of major depressive disorder.Therefore,DAPK1 may be related to cognitive dysfunction in major depressive disorder.In this study,we established a mouse model of major depressive disorder by housing mice individually and exposing them to chronic,mild,unpredictable stressors.We found that DAPK1 and tau protein levels were increased in the hippocampal CA3 area,and tau was hyperphosphorylated at Thr231,Ser262,and Ser396 in these mice.Furthermore,DAPK1 shifted from axonal expression to overexpression on the cell membrane.Exercise and treatment with the antidepressant drug citalopram decreased DAPK1 expression and tau protein phosphorylation in hippocampal tissue and improved both depressive symptoms and cognitive dysfunction.These results indicate that DAPK1 may be a potential reason and therapeutic target of cognitive dysfunction in major depressive disorder.

WJH 6^(th) Anniversary Special Issues(7): Nonalcoholic fatty liver disease Pathogenesis and therapeutic approaches for non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease affects approximately one-third of the population worldwide, and its incidence continues to increase with the increasing prevalence of other metabolic disorders such as type 2 diabetes. As non-alcoholic fatty liver disease can progress to liver cirrhosis, its treatment is attracting greater attention. The pathogenesis of non-alcoholic fatty liver disease is closely associated with insulin resistance and dyslipidemia, especially hypertriglyceridemia. Increased serum levels of free fatty acid and glucose can cause oxidative stress in the liver and peripheral tissue, leading to ectopic fat accumulation, especially in the liver. In this review, we summarize the mechanism underlying the progression of hepatic steatosis to steatohepatitis and cirrhosis. We also discuss established drugs that are already being used to treat non-alcoholic fatty liver disease, in addition to newly discovered agents, with respect to their mechanisms of drug action, focusing mainly on hepatic insulin resistance. As well, we review clinical data that demonstrate the efficacy of these drugs, together with improvements in biochemical or histological parameters.

Pathogenic and therapeutic role of exosomes in neurodegenerative disorders

Neurodegenerative disorders affect millions of people worldwide,and the prevalence of these disorders is only projected to rise as the number of people over 65 will drastically increase in the coming years.While therapies exist to aid in symptomatic relief,effective treatments that can stop or reve rse the progress of each neurodegenerative disease are lacking.Recently,research on the role of extracellular vesicles as disease markers and therapeutics has been intensively studied.Exosomes,30-150 nm in diameter,are one type of extracellular vesicles facilitating cell-to-cell communication.Exosomes are thought to play a role in disease propagation in a variety of neurodegenerative diseases,such as Alzheimer's disease,Parkinson s disease,and amyotrophic lateral sclerosis.Accordingly,the exosomes derived from the patients are an invaluable source of disease biomarkers.On the other hand,exosomes,especially those derived from stem cells,could serve as a therapeutic for these disorders,as seen by a rapid increase in clinical trials investigating the therapeutic efficacy of exosomes in different neurological diseases.This review summarizes the pathological burden and therapeutic approach of exosomes in neurodegenerative disorders.We also highlight how heat shock increases the yield of exosomes while still maintaining their therapeutic efficacy.Finally,this review concludes with outstanding questions that remain to be addressed in exosomal research.

Mesenchymal stem cell-derived extracellular vesicles as a new therapeutic strategy for ocular diseases

Mesenchymal stem cells(MSCs) have attracted considerable attention for their activity in the treatment of refractory visual disorders. Since MSCs were found to possess the beneficial effects by secreting paracrine factors rather than direct differentiation, MSC-derived extracellular vesicles(EVs) were widely studied in various disease models. MSCs generate abundant EVs, which act as important mediators by exchanging protein and genetic information between MSCs and target cells. It has been confirmed that MSC-derived EVs possess unique antiinflammatory, anti-apoptotic, tissue repairing, neuroprotective, and immunomodulatory properties, similar to their parent cells. Upon intravitreal injection, MSC-derived EVs rapidly diffuse through the retina to alleviate retinal injury or inflammation. Due to possible risks associated with MSC transplantation, such as vitreous opacity and pathological proliferation, EVs appear to be a better choice for intravitreal injection. Small size EVs can pass through biological barriers easily and their contents can be modified genetically for optimal therapeutic effect. Hence, currently, they are also explored for the possibility of serving as drug delivery vehicles. In the current review, we describe the characteristics of MSC-derived EVs briefly, comprehensively summarize their biological functions in ocular diseases, and discuss their potential applications in clinical settings.

Rationale for the use of multifunctional drugs as neuroprotective agents for glaucoma

Glaucoma, the leading cause globally of irreversible blindness, is a neurodegenerative disease characterized by progressive retinal ganglion cell death. To date, no drug has been shown to prevent the retinal ganglion cell loss associated with glaucoma. Multiple mechanisms lead to ganglion cell death in glaucoma, suggesting that a neuroprotectant that has a single mode of action, like memantine, would have a limited positive effect at slowing down ganglion cell death. Conversely simultaneously targeting several factors may be the best therapeutic approach to improve outcomes Multifunctional drugs are fast gaining acceptance as a strategy for the treatment of complex disorders of the central nervous system, such as Parkinson＇s disease, Alzheimer＇s disease and other progressive neurodegenerative diseases. In this paper, we review the current literature on multifunctional drugs and propose a rationale for the use of multifunctional drugs in glaucomatous optic neuropathy.