Nádorové buňky se od buněk normální tkáně liší mimo jiné aktivací různých signálních drah. Tyto změny jsou podmíněny odlišnou aktivitou kináz a fosfatáz způsobujících rozdíly ve fosforylaci proteinů. Detekce těchto změn je zásadní pro cílenou léčbu zaměřenou na inhibici nebo aktivaci konkrétních signálních drah. Existují různé metody, kterými lze fosforylaci proteinů stanovit. Jednou z vhodných metod schopných charakterizovat fosfoproteom je hmotnostní spektrometrie. Tento článek je zaměřen na analýzu změn ve fosfoproteomu buněčné linie MDA MB 468 odvozené od nádoru prsu s vnesenými geny kódujícími izoformy proteinu p63. Buňky s tetracyklinem indukovanou expresí izoforem proteinu p63 byly srovnány s kontrolními buňkami s přirozenou expresí p63. Denaturované proteiny z buněčného lyzátu byly nejprve enzymaticky štěpeny na peptidy a následně obohaceny o fosfopeptidy. Měření probíhalo pomocí kapalinového chromatografu spojeného s hmotnostním spektrometrem Orbitrap Elite. Každý vzorek byl analyzován třemi různými hmotnostně spektrometrickými metodami s cílem najít nejvhodnější podmínky pro detekování fosforylovaných peptidů. Z naměřených dat pak byly identifikovány a kvantifikovány fosfoproteiny. Počet fosfoproteinů identifikovaných jednotlivými hmotnostně spektrometrickými metodami byl srovnatelný, avšak každou metodou bylo získáno několik unikátních fosforylovaných proteinů. Analýzou získaných dat bylo zjištěno, že v buňkách MDA MB 468 obě izoformy proteinu p63 (TAp63α a ∆Np63α) ovlivňují zejména fosforylaci proteinů odpovědných za sestřih RNA.

Compared to normal cells, tumor cells can show different activity of kinases and phosphatases resulting in altered phosphorylation states of proteins affecting their activity within various signaling pathways. The detection of these alterations is essential for development of targeted therapy based on activation/inhibition of specific signaling pathways. Various methods can be used for detection of protein phosphorylation; however, a comprehensive assessment of phosphoproteome is performed by mass spectrometry. The differences in phosphoproteome were studied using MDA MB 468 cell line (with incorporated genes encoding isoforms of p63) derived from breast carcinoma. Cells with tetracycline‑induced expression of the p63 isoforms were compared to control cells with wild‑type expression. Denatured proteins from cell lysates were digested to peptides, enriched for phosphopeptides and subsequently separated using liquid chromatograph coupled with mass spectrometer Orbitrap Elite. Three different mass spectrometric methods were used for each sample analysis to find the most suitable conditions for the detection of phosphorylated peptides. Then phosphoproteins were identified and quantified. The number of identified phosphoproteins using all chosen mass spectrometric methods was similar; however, each method showed several unique phosphorylated proteins. Our analysis revealed that both p63 isoforms (TAp63α a ∆Np63α) mainly affected phosphorylation of proteins associated with RNA splicing in MDA MB468 cells. Key words: mass spectrometry – phosphoproteins – signaling pathways – p63 isoforms – breast cancer This study was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), by the project MEYS – NPS I – LO1413, by MH CZ – DRO (MMCI, 002 09805), by IGA NT/14602-3/2013 and BBMRI_CZ (LM2010004). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 9. 4. 2015 Accepted: 20. 7. 2015

• Klíčová slova
• izoformy p63, fosfoproteom, fragmentace,
• MeSH
• chromatografie kapalinová MeSH
• fosfopeptidy MeSH
• fosfoproteiny * analýza   MeSH
• fosforylace MeSH
• hmotnostní spektrometrie metody   statistika a číselné údaje   MeSH
• nádorové buněčné linie * MeSH
• nádorové supresorové proteiny analýza   MeSH
• nádory prsu diagnóza   MeSH
• protein - isoformy * MeSH
• proteom analýza   MeSH
• signální transdukce MeSH
• tandemová hmotnostní spektrometrie statistika a číselné údaje   MeSH
• tetracyklin diagnostické užití   MeSH
• transkripční faktory analýza   MeSH
• Publikační typ
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Východiska: Transkripční faktor p63 je homologem p53, nicméně jeho role ve vývoji a onkogenezi není tak jednoznačná. Gen TP63 kóduje množství N-a C-koncových izoforem proteinu, jejichž exprese a funkce se navzájem liší. Nejlépe prostudované jsou N-koncové varianty DNp63 (p40) a TAp63. p40 je charakteristickým markerem bazálních nebo myoepiteliálních buněk víceřadých epitelů a podílí se na regulaci proliferace a diferenciace. TAp63 izoforma je více exprimována v buňkách suprabazální vrstvy a kromě epitelů také v primárních oocytech, lymfocytech a stromálních buňkách, je schopná indukovat apoptózu a hraje roli v udržování genomové integrity. Role jednotlivých izoforem se liší také v nádorové progresi, kdy p40 je obecně považována za onkoprotein zapojený do regulace nádorových kmenových buněk, zatímco exprese TAp63 je spojována s lepší prognózou onemocnění. Cíl: Jaderná exprese p63 je markerem používaným v klinické patologii k diagnostice různých typů malignit, nejčastěji dlaždicobuněčného karcinomu plic či karcinomů urogenitálního traktu, svou roli hraje také ve vyšetření bioptických vzorků karcinomu prsu (breast carcinoma -BC) a prostaty (prostate carcinoma - PC). U neoplastických buněk lze však při použití protilátky 4A4 pozorovat expresi p63 také v cytoplazmě či v extracelulárním materiálu. Tento přehledový článek stručně shrnuje možnosti a nástrahy použití p63 v bioptické diagnostice, zejména u BC a PC, poukazuje také na potenciál využití specifických protilátek proti izoformám p40 a TAp63. Zabývá se rovněž dalším klinickým využitím p63 v histogenetické klasifikaci nádorů.

Background: The p63 transcription factor is a p53 homologue; however, its role in development and oncogenesis is more unambiguous than that of p53. TP63 encodes a variety of N-and C-terminal isoforms with different expression patterns and functions. The most frequently studied are N-terminal variants DNp63 (p40) and TAp63. p40 is a characteristic basal or myopithelial cell marker of stratified epithelium and it partakes in the regulation of proliferation and differentiation. The TAp63 isoform is more expressed in the suprabasal cell layer but is also expressed in primary oocytes, lymphocytes and stromal cells. It induces apoptosis and plays a role in the maintenance of genome integrity. The role of each isoform differs also in tumor progression. p40 is generally considered to behave as an oncoprotein in the regulation of cancer stem cells, while TAp63 expression is associated with a better prognosis. Aim: Nuclear expression of p63 is a widely used as a diagnostic marker in the clinical pathology of a spectrum of malignancies, mostly lung squamous cell carcinomas and urogenital tract carcinomas; however, it also used during examination of breast (BC) and prostate carcinoma bioptic samples. However, cytoplasmic or extracellular p63 expression is observed in neoplastic cells when the 4A4 antibody is used. This mini-review briefly summarizes the possibilities and pitfalls of p63 usage, particularly when it is used in bioptic diagnostics of BC and prostate carcinoma and highlights the potential applications of isoform specific p40 and TAp63 antibodies. It also describes other clinical usages of p63, for instance in the histogenetical classification of tumors.

• MeSH
• imunohistochemie metody   MeSH
• lidé MeSH
• nádorové proteiny fyziologie   MeSH
• nádorové supresorové proteiny * fyziologie   MeSH
• nádory prostaty * diagnóza   MeSH
• nádory prsu * diagnóza   MeSH
• nádory diagnóza   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• přehledy MeSH

Protein p63 je transkripční faktor, který má významnou funkci ve vývoji a diferenciaci epidermálních struktur a v průběhu tumorigeneze. Je členem rodiny nádorového supresoru p53 a vyskytuje se minimálně v počtu šesti izoforem, které mají během vývoje epidermis a při vzniku a progresi nádorů opačné funkce. Protein p63 ovlivňuje proliferaci a diferenciaci epidermálních buněk v průběhu ontogeneze: vrozené mutace v genu TP63 vedou k různým vývojovým deformacím a odstranění tohoto genu u myší má za následek ztrátu epidermis. Protein p63 také ovlivňuje buněčnou adhezi prostřednictvím regulace desmozomů. Ztráta kontroly proliferace buněk a mezibuněčné adheze je přitom důležitou událostí při vývoji nádorů a vysoká hladina p63 podporuje růst nádorů a brání apoptóze nádorových buněk. Tento přehledový článek stručně shrnuje úlohy proteinu p63 ve vývoji epitelů, buněčné proliferaci, adhezi a migraci a poodhaluje jeho význam při vzniku nádorových onemocnění a tvorbě metastáz.

p63 is a transcription factor which plays an important role in epidermal development, diffe­rentiation and tumourigenesis. p63 belongs to the p53 protein family and at least six isoforms were identified to date. p63 isoforms play contrary roles during the development and formation of the epidermis as well as in cancer. p63 participates in epithelial development, where it affects proliferation and differentiation of epidermal cells. Inherited mutations in the TP63 gene generate different developmental defects and p63 knockout in mice results in the absence of epidermis. Another important role of p63 is the control of cell-cell adhesion, where it regulates desmosomes. The loss of proliferation and cell-cell adhesion control are important for tumourigenesis and overexpression of p63 can enhance tumour growth and inhibit apoptosis. This review briefly summarises the roles of p63 in epithelial development, cellular proliferation, adhesion and migration and reveals its share in tumourigenesis and metastasis.

• Klíčová slova
• epidermální vývoj, vývoj nádorového onemocnění, buněčná proliferace,
• MeSH
• buněčná adheze MeSH
• buněčná diferenciace MeSH
• epidermální buňky MeSH
• epidermis * patofyziologie   MeSH
• keratinocyty cytologie   fyziologie   MeSH
• lidé MeSH
• nádorová transformace buněk MeSH
• nádorové supresorové proteiny * fyziologie   MeSH
• nádory patofyziologie   MeSH
• proliferace buněk MeSH
• proteiny regulující apoptózu MeSH
• transkripční faktory * fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

The p63 gene encodes at least six different proteins with homology to the tumour suppressor protein p53 and the related p53 family member p73. So far, there have been limited data concerning the expression patterns of individual p63 proteins, due to a lack of reagents that distinguish between the different isoforms. Three antibodies have been produced specifically directed against the two N-terminal isoforms (TAp63 and DeltaNp63) and the C-terminal region of the p63alpha proteins. TAp63 proteins are located suprabasally in stratified epithelia compared with the N-terminal truncated forms, which are more abundantly expressed in the basal cell layer, indicating a switch in expression of p63 isoforms during normal cellular differentiation. Analysis of squamous cell carcinomas shows DeltaNp63alpha to be the most widely expressed isoform, compatible with a role for this protein in promoting neoplastic cell growth in these tissues. DeltaNp63 protein expression is also restricted to basal cells in breast and prostate, whilst TAp63 isoforms are more widely expressed in these tissues as well as in tumours at these sites. TAp63, but not DeltaNp63 or p63alpha, is detected in normal colon and in colon carcinoma. TAp63 proteins are also expressed in the nuclei of a sub-population of lymphoid cells and in most malignant lymphomas, whereas DeltaNp63 proteins are not expressed. Taken together, a hitherto unrecognized regulation of p63 isoform expression in vivo has been uncovered, with different p63 proteins expressed during differentiation and in different cell types. The data indicate roles for specific p63 isoforms not only in maintaining epithelial stem cell populations, but also in cellular differentiation and neoplasia. Copyright 2002 John Wiley & Sons, Ltd.

• MeSH
• adenokarcinom genetika   metabolismus   MeSH
• antisérum imunologie   MeSH
• buněčné kultury MeSH
• DNA vazebné proteiny MeSH
• epitel metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• lidé MeSH
• lymfom genetika   metabolismus   MeSH
• membránové proteiny * MeSH
• messenger RNA genetika   MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny genetika   metabolismus   MeSH
• nádorové supresorové proteiny MeSH
• nádory genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• RNA nádorová genetika   MeSH
• spinocelulární karcinom genetika   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• transkripční faktory MeSH
• tumor supresorové geny MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, β, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. RESULTS: Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. CONCLUSIONS: In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

• MeSH
• Burkittův lymfom genetika   MeSH
• difúzní velkobuněčný B-lymfom genetika   MeSH
• genové regulační sítě genetika   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny genetika   MeSH
• protein - isoformy genetika   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• spinocelulární karcinom genetika   MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• Klíčová slova
• proximity ligation assay,
• MeSH
• fluorescenční protilátková technika MeSH
• geny p53 * MeSH
• mapování interakce mezi proteiny * MeSH
• nádorové supresorové proteiny * MeSH
• nádorový supresorový protein p53 MeSH
• nádory patologie   MeSH
• oligonukleotidové sondy MeSH
• oligonukleotidy MeSH
• protein - isoformy MeSH
• protilátky MeSH
• Publikační typ
• práce podpořená grantem MeSH

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.

• MeSH
• adenokarcinom diagnóza   imunologie   MeSH
• DNA vazebné proteiny imunologie   MeSH
• imunohistochemie metody   normy   MeSH
• jaderné proteiny imunologie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny chemie   imunologie   MeSH
• monoklonální protilátky diagnostické užití   imunologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny imunologie   MeSH
• nádorový supresorový protein p53 imunologie   MeSH
• nádory děložního čípku diagnóza   imunologie   MeSH
• nádory plic diagnóza   imunologie   MeSH
• nádory prsu diagnóza   imunologie   MeSH
• nemalobuněčný karcinom plic diagnóza   imunologie   MeSH
• protein - isoformy MeSH
• specificita protilátek imunologie   MeSH
• spinocelulární karcinom diagnóza   imunologie   MeSH
• zkřížené reakce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

The TP63 gene encodes two major protein variants that differ in their N-terminal sequences and have opposing effects. In breast, ΔNp63 is expressed by immature stem/progenitor cells and mature myoepithelial/basal cells and is a characteristic feature of basal-like triple-negative breast cancers (TNBCs). The expression and potential role of TAp63 in the mammary gland and breast cancers is less clear, partly due to the lack of studies that employ p63 isoform-specific antibodies. We used immunohistochemistry with ΔNp63-specific or TAp63-specific monoclonal antibodies to investigate p63 isoforms in 236 TNBCs. TAp63, but not ΔNp63, was seen in tumour-associated lymphocytes and other stromal cells. Tumour cells showed nuclear staining for ΔNp63 in 17% of TNBCs compared to 7.3% that were positive for TAp63. Whilst most TAp63+ tumours also contained ΔNp63+ cells, the levels of the two isoforms were independent of each other. ΔNp63 associated with metaplastic and medullary cancers, and with a basal phenotype, whereas TAp63 associated with androgen receptor, BRCA1/2 wild-type status and PTEN positivity. Despite the proposed effects of p63 on proliferation, Ki67 did not correlate with either p63 isoform, nor did they associate with p53 mutation status. ΔNp63 showed no association with patient outcomes, whereas TAp63+ patients showed fewer recurrences and improved overall survival. These findings indicate that both major p63 protein isoforms are expressed in TNBCs with different tumour characteristics, indicating distinct functional activities of p63 variants in breast cancer. Analysis of individual p63 isoforms provides additional information into TNBC biology, with TAp63 expression indicating improved prognosis.

• MeSH
• fenotyp MeSH
• fosfohydroláza PTEN genetika   MeSH
• lidé MeSH
• lokální recidiva nádoru genetika   MeSH
• mutace genetika   MeSH
• nádorové supresorové proteiny genetika   MeSH
• protein - isoformy genetika   MeSH
• protein BRCA1 genetika   MeSH
• protein BRCA2 genetika   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• transkripční faktory genetika   MeSH
• triple-negativní karcinom prsu genetika   metabolismus   mortalita   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The p53 family member p63 exists as two major protein variants (TAp63 and ΔNp63) with distinct expression patterns and functional properties. Whilst downstream target genes of p63 have been studied intensively, how p63 variants are themselves controlled has been relatively neglected. Here, we review advances in understanding ΔNp63 and TAp63 regulation, highlighting their distinct pathways. TAp63 has roles in senescence and metabolism, and in germ cell genome maintenance, where it is activated post-transcriptionally by phosphorylation cascades after DNA damage. The function and regulation of TAp63 in mesenchymal and haematopoietic cells is less clear but may involve epigenetic control through DNA methylation. ΔNp63 functions to maintain stem/progenitor cells in various epithelia and is overexpressed in squamous and certain other cancers. ΔNp63 is transcriptionally regulated through multiple enhancers in concert with chromatin modifying proteins. Many signalling pathways including growth factors, morphogens, inflammation, and the extracellular matrix influence ΔNp63 levels, with inconsistent results reported. There is also evidence for reciprocal regulation, including ΔNp63 activating its own transcription. ΔNp63 is downregulated during cell differentiation through transcriptional regulation, while post-transcriptional events cause proteasomal degradation. Throughout the review, we identify knowledge gaps and highlight discordances, providing potential explanations including cell-context and cell-matrix interactions. Identifying individual p63 variants has roles in differential diagnosis and prognosis, and understanding their regulation suggests clinically approved agents for targeting p63 that may be useful combination therapies for selected cancer patients. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

• MeSH
• lidé MeSH
• nádorové supresorové proteiny * MeSH
• nádory * MeSH
• protein - isoformy MeSH
• transkripční faktory * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

The transcription factor p63 has important functions in tumorigenesis, epidermal differentiation and stem cell self-renewal. The TP63 gene encodes multiple protein isoforms that have different or even antagonistic roles in these processes. The balance of p63 isoforms, together with the presence or absence of the other p53 family members, p73 and p53, has a striking biological impact. There is increasing evidence that interactions between p53-family members, whether cooperative or antagonistic, are involved in various cell processes. This review summarizes the current understanding of the role of p63 in tumorigenesis, metastasis, cell migration and senescence. In particular, recent data indicate important roles in adult stem cell and cancer stem cell regulation and in the response of cancer cells to therapy.

• MeSH
• kmenové buňky metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové kmenové buňky metabolismus   MeSH
• nádorové supresorové proteiny genetika   metabolismus   fyziologie   MeSH
• nádory metabolismus   MeSH
• protein - isoformy genetika   metabolismus   fyziologie   MeSH
• transkripční faktory genetika   metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH