CGB3, CGB5, CGB6, CGB7, CGB8, and CGB9, mapped to the 19q13.32 locus. This gene cluster also encompasses the CGB1 and CGB2 genes, which were originally considered to be pseudogenes, but as documented by several studies are transcriptionally active. Even though the protein products of these genes have not yet been identified, based on The Cancer Genome Atlas (TCGA) database analysis we showed that the mutual presence of CGB1 and CGB2 transcripts is a characteristic feature of cancers of different origin, including bladder urothelial carcinoma, cervical squamous cell carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, ovarian serous cystadenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, rectum adenocacinoma, testis germ cell tumors, thymoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma.Human chorionic gonadotropin (hCG) is a well-known hormone produced by the trophoblast during pregnancy as well as by both trophoblastic and non-trophoblastic tumors. hCG is built from two subunits: α (hCGα) and β (hCGβ). The hormone-specific β subunit is encoded by six allelic genes:  Human chorionic gonadotropin (hCG) is a heterodimeric hormone, which comprises two non-covalently linked subunits: alpha (α) and beta (β). The α subunit is common to all gonadotropic hormones, such as follicle stimulating hormone (FSH), thyrotropin (TSH) and luteinizing hormone (LH), while the β subunit is hormone specific and determines its biological properties ,2,3. MosHuman chorionic gonadotropin participates in and regulates many physiological processes related to the normal course of pregnancy, which includes: maintenance of progesterone production by the corpus luteum, chorionic villi development, embryo implantation, or angiogenesis . In addiHuman chorionic gonadotropin, especially its β subunit (hCGβ) is also secreted in gestational trophoblastic disease, and by a large group of non-trophoblastic tumors. hCGβ expression has been observed in tumors of different origin such as: breast, cervix, prostate, lung, colon, kidney, bladder, pancreas, anus, vulva, ovary, brain, endometrium and mouth ,9.The mechanisms regulating hCG and hCGβ expression, as well as their role in cancer development and growth, are not fully understood.CGA gene, located on the long arm of the sixth chromosome at the 6q14-q21 locus. The β subunit is encoded by eight allelic genes: CGB1, CGB2, CGB3, CGB5, CGB6, CGB7, CGB8, and CGB9, located in the luteinizing hormone beta subunit/chorionic gonadotropin beta subunit (LHB/CGB) cluster on the long arm of the nineteenth chromosome at the 19q13.32 locus [CGB4 gene encoding the β subunit of luteotropic hormone [The formation of functional human chorionic gonadotropin is associated with the activation of genes encoding both subunits of the hormone (α and β). The α subunit is encoded by a single 32 locus . The clu hormone ,12.CGB7 and CGB3 genes are allelic forms of the CGB6 and CGB9 genes, respectively [Interestingly, sequencing of the nineteenth chromosome revealed that the ectively ,14.CGB genes show amino acid differences at position 117. This led to the division of the genes into two groups. The first group, which includes the CGB7 and CGB9 genes, was categorized as type I genes and gives rise to protein products with alanine in position 117. Type II genes, which include CGB3, CGB5, CGB6, and CGB8, encode a protein with aspartic acid in position 117 [The protein products of individual tion 117 ,12,15.CGB3, CGB5, CGB6, CGB7, CGB8, and CGB9 genes arose as a result of multiple duplications of the gene encoding the β subunit of luteinizing hormone (CGB4) [CGB1 and CGB2 genes evolved as a consequence of the insertion of a DNA fragment into the duplicated genes. In the case of the CGB1 gene, this fragment included 736 base pairs, while for CGB2, it was 724 base pairs. The insertion led to the deletion of a 52-base long segment of the proximal promoter, as well as the entire 5′ untranslated region (5′UTR) region of the CGB gene. The consequence of this mutation was the creation of a new promoter sequence for CGB1 and CGB2, a new 5′UTR region with an alternative start codon, and a new first exon. This led to a shift in the open reading frame for exons 2 and 3 [CGB1 and CGB2 confirms their common evolutionary path of origin [It is hypothesized that in the course of evolution, the e (CGB4) . In cont 2 and 3 ,17. The f origin .CGB1 and CGB2 were considered to be pseudogenes. Their biological activity was, however, demonstrated at the level of transcription. The genes’ transcripts have been detected in healthy tissues, such as placenta, testes and the pituitary gland [CGB1 and CGB2 expression has also been confirmed in breast and bladder cancer cell lines, as well as in ovarian cancer tissue [Until recently, ry gland ,20,21,22r tissue ,23,24,25CGB1 and CGB2 in the total expression of all CGB genes does not exceed 1/1000, both CGB1 and CGB2 genes were shown to have a considerable expression peak during the first trimester in normal pregnancy, but not in placentas of the second and third trimester, or in recurrent miscarriages. This particular expression pattern of the genes suggests their putative role in the implantation stage of the fetus development. Thus, it may be speculated that their expression drives placental cytotrophoblast cell invasion and growth during implantation. In the same manner, CGB1 and CGB2 may promote the pregnancy-like invasion of cancer cells into specific microenvironments, and later tumor growth and metastasis [Although the contribution of tastasis .CGB1 and CGB2 genes in cancer, we analyzed data collected in TCGA.In order to verify the transcriptional activity of CGB1 and CGB2 gene expression. Twelve tumors, characterized by the presence of both CGB1 and CGB2 gene transcripts, were selected for further research and the expression level of the studied genes in cancer and corresponding non-malignant tissues was analyzed using UALCAN web-portal [Information related to thirty-three different types of tumors deposited in The Cancer Genome Atlas (TCGA) database was evaluated in terms of b-portal .CGB1 and CGB2 in non-malignant and primary tumor samples for each gene was presented as a box and whisker plot. All values of gene expression were presented as transcripts per million (TPM)—a normalization method for RNA-seq which represents the relative abundance of a gene or transcript in a sample.TCGA level 3 RNASeq V2 data equivalent to the expression of t-test was used to calculate the level of statistical significance . In the case of a lack of information regarding the expression of the genes or when the number of available data was extremely small, no statistical test was performed.Student’s The data were presented using figures showing the interquartile range (IQR), including the median, minimum, maximum values, 25th percentile (Lower Q) and 75th (Upper Q) percentile (if available).CGB1 and CGB2 genes in cancers of different origin, the molecular characterization of thirty-three different tumors deposited in the TCGA database was analyzed. First, the screening of TCGA transcriptome data related to CGB1 and CGB2 gene expression in different cancers was performed.To evaluate the expression level of CGB1 gene transcript was found in 12 types of cancer only, namely: BLCA , CESC , ESCA , HNSC (Head and Neck squamous cell carcinoma), OV (Ovarian serous cystadenocarcinoma), LUSC (Lung squamous cell carcinoma), PAAD (Pancreatic adenocarcinoma), READ (Rectum adenocacinoma), TGCT (Testis germ cell tumors), THYM (Thymoma), UCEC  and USC (Uterine carcinosarcoma);  (The arcoma); . The higctively) .CGB2 gene transcripts were found in 26 types of cancer: ACC , BLCA , BRCA (Breast invasive carcinoma), CESC , CHOL (Cholangiocarcinoma), DLBC (Lymphiod neoplasm diffuse large B-cell lymphoma), ESCA , HNSC (Head and Neck squamous cell carcinoma), KIRC , LGG (Brain lower grade glioma); OV (Ovarian serous cystadenocarcinoma), MESO (Mesothelioma), LUAD (Lung adenocarcinoma), LUSC (Lung squamous cell carcinoma), PAAD (Pancreatic adenocarcinoma); PRAD (Prostate adenocarcinoma), PCPG (Pheochromocytoma and Paraganglioma), READ (Rectum adenocacinoma), SARC (Sarcoma), SKCM (Skin cutaneous melanoma); TGCT (Testis germ cell tumors), THCA (Thyroid carcinoma), THYM (Thymoma), STAD (Stomach adenocarcinomna), UCEC  and USC (Uterine carcinosarcoma); (The arcoma); .CGB2 transcriptional activity were greater than 0, ; ; .CGB1 and CGB2. This group consisted of BLCA, CESC, ESCA, HNSC, OV, LUSC, PAAD, READ, TGCT, THYM, UCEC and USC (CGB genes (CGB1: 3.158 and CGB2: 4.154) characterized UCEC  (Significant differences in  0.0044) .CGB1–CGB9. The expression level of individual CGB genes is not equal. The most transcriptionally active gene, with up to twenty times higher expression compared to other CGB genes in both placenta and many cancerous tissues, is CGB5. The genes with lower transcriptional activity are CGB3, CGB6, CGB7, CGB8, and CGB9, as well as CGB1 and CGB2 [The synthesis of the β subunit of human chorionic gonadotropin occurs as a result of the activity of the tissue-dependent genes mapped to chromosome 19 (19q13.3), called and CGB2 .CGB1 and CGB2 transcripts in cells and tissues. In fact, for this reason, the CGB1 and CGB2 genes were considered pseudogenes.For a long time, the methods used for quantifying gene expression were insufficiently sensitive to detect the presence of CGB1 and CGB2 are a thousand times lower compared to the remaining CGBs, it has been shown that these genes’ transcripts play an important role in chorionic villi invasion and embryo implantation [Although the expression levels of antation . It was CGB2 expression, but not other CGB genes, which correlates with the amount of functional free hCGβ protein secreted by cancer cells and with cancer growth. It was also demonstrated that specific targeting of CGB1 and CGB2 with siRNA was much more effective in reducing cancer cell numbers than silencing other CGB genes [CGB1 and CGB2 products give rise to tumor-driving molecules.Indeed, it was reported that in bladder cancer cell lines, it is GB genes . Thus, iCGB1 and CGB2 in cancer was mainly shown in cell lines, the aim of the present study was to demonstrate the expression of these genes in human cancer tissues of different origin. The data deposited in TCGA database was used in order to verify CGB1 and CGB2 transcriptional activity status in malignant and corresponding non-malignant tissue.Since the transcriptional activity of CGB1 transcriptional activity characterized twelve types of cancer only, namely: BLCA, CESC, ESCA, HNSC, OV, LUSC, PAAD, READ, TGCT, THYM, UCEC, and USC.The results of the study showed that out of thirty-three available records related to the studied genes’ expression, CGB2 transcripts were detected in twenty six cancers, including: ACC, BLCA, BRCA, CESC, CHOL, DLBC, ESCA, HNSC, KIRC, LGG, OV, MESO, LUAD, LUSC, PAAD, PRAD, PCPG, READ, SARC, SKCM, TGCT, THCA, THYM, STAD, UCEC, and USC.At the same time CGB1 and CGB2. This group included bladder urothelial carcinoma, cervical squamous cell carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, ovarian serous cystadenocarcinoma, lung squamous cell carcinoma, pancreatic adenocarcinoma, rectum adenocacinoma, testis germ cell tumors, thymoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma.Interestingly, only twelve cancer types were characterized by the concomitant expression of CGB1 and CGB2 genes in cancerous tissues of different origin was confirmed. Significant differences between CGB2 expression levels in healthy and cancerous tissues were shown. Such differences, which characterized bladder urothelial carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, lung squamous cell carcinoma, and uterine corpus endometrial carcinoma, may have diagnostic implications. In the case of the CGB1 gene, such a difference was recognized for uterine corpus endometrial carcinoma only. The analysis of the effect of CGB1 and CGB2 gene expression on patient survival did not show any significant correlation.Despite the fact that the studied genes’ expression levels in cancer tissues were low, with the median often equaling zero, the transcriptional activity of the CGB1 and CGB2 gene products are believed to drive placental cytotrophoblast cell invasion and growth during implantation. In the same way, they may promote the pregnancy-like invasion of cancer cells into specific microenvironments, and later tumor growth and metastasis. In fact, the results of TCGA database analysis show that a crucial factor in carcinogenesis may be CGB2 gene expression. The gene’s products were detected in 26 out of 33 studied samples, and statistically significant differences in CGB2 expression between malignant and non-malignant tissues were noted.As mentioned earlier, CGB1 and CGB2 gene products drive tumor development.However, taking into account the limited data available, it cannot be excluded that both CGB1 and CGB2 genes are involved in tumor growth, invasion and metastasis.Further studies are needed in order to verify the hypothesis that


This strategy hinges on the availability of appropriate, validated collection and transport systems to ensure preservation of nucleic acids and compatibility with downstream molecular testing—an acute challenge in the current pandemic. We present direct comparison of COVID-19 specimens collected with FLOQSwab nasopharyngeal swab preserved in universal transport medium , optimized for viral specimens, and flocked regular nylon tip swab preserved in liquid Amies , optimized for bacterial specimens.On 16 March 2020, the WHO Director-General stated, “You cannot fight a fire blindfolded. And we cannot stop this [COVID-19] pandemic if we don’t know who is infected. We have a simple message for all countries: test, test, test. Test every suspected case”  amplification platforms. In the first, nucleic acid extraction/amplification was performed on the BD Max System , using the ExK TNA-2 extraction strip and detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 5′ untranslated region (UTR). Alternatively, specimens were extracted on the NucliSENS EasyMAG  and detection of SARS-CoV-2 5′ UTR and envelope (TC) values for each of the 3 rRT-PCR targets (5′ UTR in BD Max and 5′ UTR and envelope gene in Rotor-gene) did not show statistically significant difference with a 2-sided paired-sample t test between the 2 collection devices , 33 were concordantly positive, 59 were concordantly negative, and 1 was only FLOQSwab positive. Positive and negative results were concordant between the 2 assays. Comparing swabs, positive percent agreement, negative percent agreement, and Cohen’s kappa values were 100% , 100% , and 1.00, respectively, on the BD Max and 97.1% , 100% , and 0.98, respectively, on the Rotor-gene. For positive swabs, the average cycle threshold . Second, detection of respiratory pathogens other than SARS-CoV-2 was not evaluated. Despite these limitations, this work clearly demonstrates, using two distinct downstream molecular testing methods, that the ESwab collection device is a suitable alternative to the UTM collection system in the context of an international swab shortage. We call upon the microbiology community to innovate and enhance testing capacity crucial to limit the spread of the current COVID-19 pandemic.


Endometrial cancer is one of the most frequently diagnosed gynecological malignancies worldwide. However, its prognosis in advanced stages is poor, and there are only few available treatment options when it recurs. Epigenetic changes in gene function, such as DNA methylation, histone modification, and non-coding RNA, have been studied for the last two decades. Epigenetic dysregulation is often reported in the development and progression of various cancers. Recently, epigenetic changes in endometrial cancer have also been discussed. In this review, we give the main points of the role of DNA methylation and histone modification in endometrial cancer, the diagnostic tools to determine these modifications, and inhibitors targeting epigenetic regulators that are currently in preclinical studies and clinical trials. POLE ultramutated, microsatellite instability (MSI) hypermutated, copy-number low, and copy-number high. Although the POLE ultramutated group (7%) has good prognosis, the copy-number high (serous-like cancers) group has poor prognosis ), the MYST family , and the p300/CBP family (p300 and CBP) . There a+-dependent, whereas class III HDACs are NAD+-dependent [Histone deacetylases are generally associated with gene silencing and transcriptional repression. HDACs also catalyze both histone and non-histone proteins . In humaependent ,71.There are only a few reports about HATs in EC. Yi et al.  showed tIn contrast to HATs, HDACs have been reported extensively in EC. HDACs regulate many cellular functions, including cell proliferation, apoptosis, and differentiation. Consequently, deregulation of HDACs is involved in tumorigenesis, therefore, HDAC inhibitors are effective in various types of cancers. In EC, HDACs are overexpressed compared to the normal endometrium ,73,74. THistone methylation occurs through the transfer of methyl groups to the arginine or lysine residues in histone tails from S-adenosylmethionine (SAM) as a methyl donor. Similar to histone acetylation, histone methylation is regulated through the addition of methyl groups by histone methyltransferases (HMTs are referred to as “writers”) and the removal of methyl groups by histone demethylases (HDMs are referred to as “erasers”). Lysine residues can be mono-, di-, or tri-methylated , whereas arginine residues can be mono-, symmetrically or asymmetrically di-methylated . Compared to histone acetylation, which promotes gene transcription, gene transcription regulated by histone methylation is more diverse and depends on the state of histone methylation ,18,78. FUnlike histone acetylation, which promotes gene transcription, histone methylation regulates both active gene transcription and gene repression. At promoter sites, H3K4me3 is associated with active genes, whereas H3K9me3 is associated with silent genes. At enhancer sites, H3K4me1 is associated with active genes. Appropriate histone methylation is critical for regulating gene expression ,18.HMTs can be divided into two groups: histone lysine methyltransferases (HKMTs) and protein arginine methyltransferases (PRMTs). HMTs also catalyze methyl transfer not only in histone proteins but also in non-histone proteins ,79,80. TMany studies have reported that histone methylation by HKMTs is linked to cancer development. Over 100 KMTs have already been identified in the human genome. Lysine methylation occurs mainly on histone H3 or H4. HKMTs can be classified into six groups according to the major histone methylation sites: H3K4 is methylated by SETD1A, SETD1B, MLL1, MLL2, MLL3, MLL4, SETD7, and PRDM9; H3K9 is methylated by SUV39H1, SUV39H2, SETDB1, and G9a, also known as EHMT2, and GLP, also known as EHMT1; H3K36 is methylated by SETD2, NSD1, NSD2, NSD3, and ASH1L; H3K79 is methylated by DOT1L; H4K20 is methylated by SET8, SUV420H1, and SUV420H2; and H3K27 is methylated by EZH1 and EZH2. In addition to these sites and enzymes, there are several other lysine methylation sites, which are not well studied, such as H3K23me3 and H3K63me3 [In humans, nine PRMTs have already been identified. Several studies report that arginine methylation by PRMTs is involved in tumorigenesis through gene transcription, X chromosome inactivation, DNA repair, and mRNA splicing . Based oHistone lysine demethylases (HDMs) can be divided into two groups: the amine-oxidase type lysine-specific demethylases (LSDs), including LSD1, also known as KDM1A, and LSD2, also known as KDM1B, and the highly conserved JumonjiC (JMJC) domain-containing histone demethylases, including JHDM1, JHDM2 (JMJD1), JHMD3 (JMJD2), JARID, PHF, and UT families . JMJD6, Aberrant histone modification is often associated with various cancers ,80. QinghMLH1 and inducing apoptosis [There have been several preclinical studies of DNA methylation inhibitors for patients with EC; however, are still no clinical studies for this class of drugs in humans . This sepoptosis . Furtherpoptosis . This supoptosis ,100,101.ClinicalTrials.gov identifier NCT03018249) that evaluates the effect of a combination HDAC inhibitors, Entinostat (MS-275) and medroxyprogesterone acetate therapy , valproic acid (VPA), and carboxylic acids NaB; hydroxamic acids, including trichostatin A (TSA) and suberoylanilide hydroxamic acid ; benzamides, such as entinostat (MS-275) and mocetinostat MGCD-0103); and cyclic peptides, such as romidepsin (FK228). In addition to these four groups, an increasing number of HDAC inhibitors have been developed ,62,102. ; and cyc therapy . SeveralClinicalTrials.gov identifier NCT04611139) has been started in October 2020. This is a study of the combination therapy of the LSD inhibitor (SP-2577) and the anti PD-1 antibody pembrolizumab in patients with EC  for EC ( with EC . Since tThe existence of H3K9 methyltransferase and EHMT2 (G9a) inhibitors, have also been reported. Here, the addition of an EMHT2 inhibitor to EC cell lines resulted in the suppression of cell proliferation in vitro as well as myometrial invasion by repressing E-cadherin .The LSD1 inhibitor, HCI2509, suppressed cell growth in type II EC cell lines and mouse xenograft models, inducing apoptosis. Elevation of H3K27me3 was also observed using an LSD1 inhibitor .The analysis considering histone modification was not carried out in each report. In fact, mechanisms that regulate downstream genes via acetylation and methylation are conceivable. It is therefore necessary to understand the detailed antitumor mechanism of these inhibitors when considering their potential clinical applications. Analysis of histone modifications, such as ChIP-seq and ATAC-seq, should be performed in the future.Epigenetic mechanisms are critical in normal biological function as well as tumorigenesis. In the past 20 years, epigenetics research has made remarkable progress, and new analyses such as ATAC-seq and ChIP-seq have also been discovered. However, there are few studies regarding the epigenome in EC. Therefore, it is necessary to deepen our understanding of epigenetic mechanisms so it could be useful in the development of therapeutic drugs and the expansion of indications of current anticancer drugs to include EC. However, there are some problems that need to be overcome. First, the epigenetic mechanisms in EC are still unknown. Even though DNA methylation has been extensively studied, there are still few reports regarding histone modification. As we have previously mentioned here, further analyses, such as ChIP-seq and ATAC-seq in EC, are needed to identify new downstream genes and develop novel biomarkers. Second, it is also important to elucidate the epigenetic mechanisms in the four molecular prognostic categories proposed by the TCGA. Although DNA methylation data from the four TCGA categories has already been revealed, to date, there are no specific reports regarding histone modifications. These aspects could help the development of novel therapeutic drugs. Although there are only two inhibitors targeting the epigenome for EC currently in clinical trials, it would be possible to expand the therapeutic potential of not only a single agent but also combination therapies with existing drugs. Although further preclinical studies and evaluation of adverse events for epigenetic inhibitors will be needed, there is hope in discovering new drugs targeting the epigenome in EC.


Background: Abnormal extracellular matrix (ECM) remodeling plays an essential role in urothelial carcinoma (UC) invasiveness and metastasis. Focusing on the ECM structural constituent (GO: 0005201), we recognized a significant upregulation of the fibulin 2 gene (FBLN2) during UC progression in a published UC transcriptome (GSE31684). Thus, we aimed to investigate the roles of FBLN2 expression and its prognostic value in upper urinary tract UC (UTUC) and urinary bladder UC (UBUC) in our large, well-characterized cohort.Patients and Methods: Clinicopathological data and formalin-fixed paraffin-embedded UC tissues were analyzed retrospectively. We determined FBLN2 expression using immunohistochemical staining assessed by H-scores. FBLN2 expression correlated with clinicopathological features and patient outcomes, including metastasis-free survival (MFS) and disease-specific survival (DSS). Statistical analyses were performed using Pearson's chi-square test, Kaplan–Meier estimates of DSS and MFS, and the Cox proportional hazards model. We used Ingenuity Pathway Analysis (IPA) to clarify the functional significance of dysregulated FBLN2 in UC.Results: Data from 295 UBUC and 340 UTUC patients were available for the final evaluation. Pearson's chi-square test showed that high FBLN2 immunoexpression significantly correlated with adverse pathologic variables, such as advanced pathologic tumor stage, high histological grade, perineural invasion, vascular invasion, lymph node metastasis, and increased mitotic rate . Kaplan–Meier analysis demonstrated associations of high FBLN2 expression with worse DSS (p < 0.001) and MFS (p < 0.001). Furthermore, multivariate analysis identified high FBLN2 expression as an independent predictive risk factor for DSS  and MFS . IPA demonstrated that multiple signaling pathways were enriched, including the oxidative phosphorylation, mitochondrial dysfunction, and regulation of the epithelial–mesenchymal transition pathways.Conclusion: High FBLN2 expression was associated with adverse pathologic features and worse oncological outcomes and may serve as a prognostic biomarker for UC. Urothelial carcinoma (UC) is a common urinary tract malignancy worldwide , 2. It mFor UBUC, ~75% of patients are initially diagnosed with non-muscle-invasive bladder cancer (NMIBC) while the remaining 25% are diagnosed with muscle-invasive bladder cancer (MIBC) , 4. TranUC tumor heterogeneity occurs at multiple levels and directly affects clinical care and treatment efficacy . PatientFBLN2) was significantly associated with advanced pathologic stage and tumor metastasis in UBUC, suggesting its role in cancer progression.To identify transcripts that are potentially involved in UC invasiveness and metastasis, we performed data mining and processing of a public transcriptome dataset, focusing on the ECM structural constituent. We found that high transcript expression of fibulin 2 -like domains and a unique C-terminal fibulin-type module   –20. Untip < 0.01 and log2 ratio >0.3) for further study. From the Oncomine database, we selected two studies, Sanchez-Carbayo et al. (FBLN2 in UBUC.A transcriptome dataset (GSE31684), including 93 UBUC patients who received radical surgery, was collected from the NCBI GEO database. To quantify the gene expression level, we analyzed all probe sets without preselection or filtering and imported the raw files into Nexus Expression 3 software . Under supervision, comparative analyses were conducted to discover the significantly differentially expressed genes related to the ECM structural constituent (GO:0005201) by comparing tumor stage (high stage vs. low stage) and metastatic events (metastasis vs. non-metastasis). We chose differentially expressed genes  for 1 h. Primary antibodies were detected using a ChemMate EnVisionTM Detection kit . Sections processed without the primary anti-FBLN2 antibody were used as negative controls. As for positive controls, we included HT 1197 cell block sections known to express high FBLN2. Two independent pathologists quantified FBLN2 protein expression levels by integrating the percentage and intensity of immunostaining in the cytoplasm of UC cells to generate the H-score using the formula ΣPi(i +1), in which Pi represents the percentage of stained tumor cells (0 to 100%) and i is the intensity of immunoreactivity (0 to 3+). The immunoreactivity was divided into low and high expression levels based on the median H-score.Four-micrometer-thick sections from formalin-fixed, paraffin-embedded blocks were sectioned on pre-coated slides followed by deparaffinization, rehydration, and antigen retrieval as previously described , 26. NexFBLN2 in UC, we used Ingenuity Pathway Analysis  to analyze ingenuity canonical pathways, upstream regulators, diseases and biological functions, and gene networks. These core analyses were used to interpret the common differentially expressed genes between high FBLN2-expressing and low FBLN2-expressing UCs.The RNA sequencing and clinical data were downloaded from the TCGA BLCA  database through the c-BioPortal platform. To understand the functional significance of dysregulated FBLN2 expression and various clinicopathological features was assessed using Pearson's chi-square test. We analyzed two end points, including metastasis-free survival (MFS) and disease-specific survival (DSS). Univariate and multivariate analyses were used to identify relevant FBLN2 expression and clinicopathological characteristics among the predictors of DSS, measured from curative surgery to the time of cancer death, and MFS, measured from curative surgery to the first metastasis. Survival curves were drawn using the Kaplan–Meier method with a log-rank test. All significant parameters for univariate analysis were included in the multivariate Cox proportional hazards model to identify the independent predictors. SPSS Statistics V.17.0 software  was utilized for statistical analyses. Statistical significance was set at p < 0.05.The association between FBLN2 for further study, as the role of FBLN2 in cancer development is not straightforward; it may inhibit or promote tumorigenesis. Its function in UC is not well-elucidated. To further confirm the initial observations, we examined the Oncomine database for FBLN2 expression in bladder cancers. The Oncomine database revealed that there was a significantly higher expression of FBLN2 in infiltrating UBUC compared to superficial tumors (FBLN2 in our well-characterized UTUC and UBUC cohorts.We utilized a published UBUC transcriptome dataset (GSE31684) for data mining, including 93 patients undergoing radical cystectomy. There were 78 patients with muscle-invasive disease and 28 patients with lymph node metastasis. Through transcriptomic profiling, we identified 13 probes covering 11 transcripts associated with the ECM structural constituent (GO: 0005201). These genes were significantly upregulated in the high pathologic stage and lymph node metastasis . We selel tumors , 2. ThesWe included 340 UTUC and 295 UBUC patients with a mean age of 65.8 years . In the In the UBUC cohort, 123 patients (41.7%) had MIBC (pT2-4), and most patients (81%) had high histological grade tumors at initial diagnosis. Concerning lymph node status, 266 patients (92.2%) were N0 and 29 (7.8%) were N1–2. There were 156 lesions (52.9%) with high mitosis. Moreover, VI and PNI were observed in 20 (6.8%) and 49 tumors (16.6%), respectively.FBLN2 immunoreactivity from low stage UC to high stage UC . Similarly, in UBUC patients, we found significant associations between high FBLN2 expression and advanced pT status (p < 0.001), high histological tumor grade (p = 0.003), lymph node metastasis (p = 0.001), VI (p < 0.001), PNI (p = 0.001), and high mitotic rate (p < 0.001).Immunohistochemical staining showed stepwise increments in stage UC . Table 2FBLN2 staining on cancer metastasis and patient death in UC.There were 113 patients, including 61 UTUCs and 52 UBUCs, who died of UC. In addition, 146 patients, including 70 UTUCs and 76 UBUCs, had subsequent metastatic tumor development. Univariate and multivariate analyses were performed to assess the prognostic impacts of FBLN2-expressing tumors died of the disease, whereas only 6.5% of patients with low FBLN2-expressing tumors did. Furthermore, 32.9% of high FBLN2-expressing tumors developed metastasis, whereas only 8.2% of the tumors with low FBLN2-expressing tumors did. Kaplan–Meier analysis showed a significant association of high FBLN2 expression with worse DSS (p = 0.0001) and worse MFS (p < 0.0001). Apart from FBLN2 immunoexpression, we found that tumor location, multifocal tumors pathologic stage, lymph node metastasis, histological grade, PNI, and VI were significant prognosticators of MFS and DSS in the univariate analysis. Multivariate Cox regression analysis showed that FBLN2 expression was independently predictive for DSS  and MFS .In UTUC , 29.4% oFBLN2-expressing tumors, 39.9% had tumor metastasis and 27.0% died of the disease, whereas only 11.6% of patients with low FBLN2-expressing tumors had subsequent metastatic tumors and 8.2% experienced cancer deaths. Following univariate analysis, we observed that advanced pT status, lymph node metastasis, high histological grade, VI, PNI, high mitotic activity, and high FBLN2 expression (p < 0.0001) were associated with worse DSS and MFS. In the multivariate analysis, FBLN2 expression status , pathologic stage, and mitotic activity were markedly associated with DSS. In addition, high FBLN2 expression , high pathologic stage, and high mitotic activity significantly correlated with worse MFS.In UBUC  patientsFBLN2 expression in TCGA BLCA. Detailed information on the 400 differentially expressed genes is provided in FBLN2 in UC, these genes were analyzed by IPA. The data demonstrate the enrichment conditions of differentially expressed genes in canonical signaling pathways. Multiple signaling pathways were enriched, including oxidative phosphorylation, mitochondrial dysfunction, sirtuin signaling pathway, and regulation of the epithelial–mesenchymal transition pathway. The IPA results identified CCR2, TGFB1, and TWIST1 among the top upstream regulators.The selection criteria of the differentially expressed genes included the top 200 most significant positively and negatively correlated genes to FBLN2 may induce the alteration of numerous biological functions and diseases, such as cancer, organismal injury and abnormalities, connective tissue disorders, tissue development and cellular assembly and organization, and cellular function and maintenance. Subsequently, the differentially expressed genes were categorized into 28 networks. The top 3 networks had higher scores and more than 25 focused molecules. Genes encoding the collagen family proteins, including COL10A1, COL14A1, COL16A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL8A1, COL8A2, OL1A2, and COL3A1, along with 13 other dysregulated genes were involved in diseases and functions related to cancer, connective tissue disorders, and organismal injury and abnormalities, whereas 25 dysregulated genes, including ADAM12, ADAMTS10, and ADAMTS2, were involved in diseases and functions related to connective tissue disorders, hereditary disorders, and organismal injury and abnormalities. In addition, 9 nuclear genes encoding mammalian mitochondrial ribosomal proteins and 20 other dysregulated genes were involved in diseases and functions associated with metabolic diseases, organismal injury and abnormalities, and protein synthesis.Additionally, in the disease and functional enrichment analyses, we identified that UC is a common and heterogeneous malignancy in the urinary system. Until recently, treatment for UC had seen little progress, and survival rates did not improve significantly over the last three decades. Therefore, the discovery of novel prognostic biomarkers remains an urgent concern in order to improve the clinical outcomes of UC patients. With advances in high-throughput technologies, many scholars have investigated the gene expression levels of UC samples and uploaded these data to public databases, such as GEO and TCGA , 28. TheFBLN2 was a significantly upregulated gene related to the ECM structural constituent and associated with advanced tumor stage and metastatic disease in UBUC. The Oncomine database also revealed that infiltrating UBUC expressed significantly higher FBLN2 than superficial tumors. Furthermore, the prognostic impact of FBLN2 immunoexpression was validated in our well-characterized UC cohort. We demonstrated that FBLN2 expression status was independently predictive for both MFS and DSS in UTUC and UBUC. In addition, high FBLN2 expression was significantly associated with adverse pathologic tumor characteristics, such as advanced pathologic tumor stage, high histological tumor grade, lymph node metastasis, VI, PNI, and high mitotic rate. These results indicate that FBLN2 plays a key role in UC development and progression. Thus, incorporation of FBLN2 immunoexpression with standard pathologic and prognostic parameters may improve UC risk stratification.Using a public domain dataset (GSE31684), we demonstrated that FBLN2 can form homodimeric complexes and bind to many ECM components, such as integrin, fibronectin, and laminin, to stabilize the organization of ECM structures  can be found in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB10501-005) of Chi Mei Medical Center. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.W-ML, Y-LS, and C-FL: study concept and design. T-CC, SH, P-IL, and Y-CW: acquisition of data. H-LK and C-FL: analysis and interpretation of data. W-ML, Y-LS, and C-FL: drafting of the manuscript. Y-LS and C-FL: critical revision of the manuscript for important intellectual content. W-ML and C-FL: statistical analysis. All authors agreed to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer’s disease (AD) is the most common type of dementia leading to progressive memory loss and cognitive impairment. Considering that pharmacological treatment options for AD are few and not satisfactory, increasing attention is being paid to dietary components that may affect the development of the disease. Such a dietary component may be caffeine contained in coffee, tea or energy drinks. Although epidemiological data suggest that caffeine intake may counteract the development of cognitive impairment, results of those studies are not conclusive. The aim of the present study is to review the existing experimental studies on the efficacy of caffeine against AD and AD-related cognitive impairment, focusing on the proposed protective mechanisms of action. In conclusion, the reports of studies on experimental AD models generally supported the notion that caffeine may exert some beneficial effects in AD. However, further studies are necessary to elucidate the role of caffeine in the effects of its sources on cognition and possibly AD risk. Alzheimer’s disease (AD) is the most common type of dementia, accounting for 50–70% of neurodegenerative dementia cases. AD leads to a progressive loss of memory and cognitive abilities .It is believed, especially taking into account very limited therapeutic options, that dietary interventions or nutraceuticals may be promising in the prophylaxis and treatment of cognitive impairment of ageing, including AD ,4,5,6,7.Coffea L.) beans, cola (Cola acuminata (P. Beauv.) Schott and Endl.) nuts, tea (Camellia sinensis (L.) Kuntze) leaves and yerba mate (Ilex paraguariensis A.St.-Hil.) leaves, as well as guarana (Paullinia cupana Kunth) seeds and cocoa (Theobroma cacao L.) beans [Caffeine  is a purine alkaloid, commonly consumed on a daily basis. Caffeine is probably the most commonly used psychoactive substance/psychostimulant ,9,10. It.) beans ,10. The .) beans . Caffein.) beans . The mea.) beans . Accordi.) beans ,14.Numerous health-promoting activities are attributed to coffee and caffeine. A recent umbrella review of meta-analyses indicated, among others, beneficial role of coffee in reducing risk of type 2 diabetes mellitus (T2D), cardiovascular diseases, some cancers and Parkinson’s disease ,16. The Here we would like to focus on the results of experimental studies of caffeine, carried out in order to evaluate its potential in the prophylaxis and treatment of AD. A literature search was conducted on the PubMed electronic database. Articles presented in languages other than English were excluded. Results of all studies on caffeine effects in different AD experimental models, published until December 2020, and found in the PubMed electronic database, were referred to in the present review.1 and A2A), demonstrating a structural similarity to adenosine. The blocking of these receptors modulates glutamatergic, cholinergic, dopaminergic, serotoninergic and noradrenergic neurotransmission [2+ from the endoplasmic reticulum [A) receptors [The mechanism of action of caffeine is complex. Caffeine is a non-selective antagonist of adenosine receptors  and sporadic. Familial AD (FAD), 5% of AD cases ,37, is cThe most popular hypothesis on the pathogenesis of AD is the amyloid hypothesis, which assumes an important role of Aβ deposits in the form of senile plaques ,49. In fAβ is formed as a result of incorrect fragmentation of the amyloid precursor protein (APP) processed by β-secretases (β-site amyloid-precursor-protein-cleaving enzyme-1—BACE-1 and BACE-2) and γ-secretases . Senile 1 muscarinic receptors (the level of presynaptic M2 receptors is decreased) in the cerebral cortex, their functions are impaired. Also, progressive decrease in the number of nicotinic receptors in the cerebral cortex is observed. Deficits in cholinergic neurotransmission contribute to the learning, memory and attention impairment [2+-dependent neuroprotection [2 receptor activation or β receptor blockade) are involved in the Aβ-related neuropathology. Upregulation of adenosine receptors (A1R and A2AR) is also observed in AD. Activation of adenosine receptors affects synaptic neurotransmission and various neurotransmitters release  [Neurodegenerative disorders in AD lead to decreases in the levels of neurotransmitters. An important role in the pathogenesis of AD is a reduction of acetylcholine (ACh) content. The dysfunction of cholinergic system is related to degeneration of the nucleus basalis of Meynert cholinergic neurons caused by NFTs formation. The presence of NFTs and Aβ plaques leads to cholinergic synapse loss. Moreover, despite the preservation of postsynaptic Mpairment ,56. Moreotection ,58. In Aotection ,60. Malfotection . Moreoveotection ,56. Alsoutamate) . Also, dutamate) .Summing up, cholinergic and glutamatergic transmission disorders are associated with impaired cognitive processes, while disorders of dopaminergic, serotonergic and noradrenergic transmission are responsible for the symptoms associated with dementia, such as depression, apathy, anxiety and psychotic symptoms .Epidemiological studies demonstrated higher occurrence of AD (2:1 women to men ratio) and risk of developing AD in women compared to men. It is probably related to the longer life expectancy for women. In addition, faster cognitive decline and longer life span after diagnosis of mild cognitive impairment or AD dementia is observed in elderly women in comparison to men ,65,66. MThe treatment options of AD are very limited. Currently, only four drugs are approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA) for AD treatment. These include three drugs from the group of acetylcholinesterase (AChE) inhibitors  and memantine, a selective and non-competitive NMDA receptor antagonist ,69. AChETaking into account the postulated pathomechanism of AD, it is believed that antioxidant nutraceuticals may have beneficial effects in the prevention of AD. The best-known antioxidants include plant polyphenols such as curcumin, flavonoids and phenolic acids. Experimental studies and/or population studies suggest the possibility of beneficial effects of curcumin ,72,73,74The studies on the effects of caffeine consumption  on the cognitive impairment, mild cognitive impairment, dementia and AD led to differential conclusions. Although the review articles and meta-analyses indicated rather favorable effects of caffeine-containing dietary sources on those disorders, their conclusions were not unequivocal ,88,89,90It should be emphasized that other than caffeine active constituents of coffee or tea  may contribute to their effects on cognition/AD risk ,93. DecaIt is crucial to use experimental models faithfully mimicking the pathologies of investigated diseases. In case of AD, it is problematic. None of the experimental AD animal models fully reflects complete disorders and cognitive impairments characteristic for human AD. Although FAD accounts for about 5% of all disease cases only, the majority of potential therapies has been investigated in transgenic mouse models of AD .Transgenic technologies allowed mouse and rat models to be created, based on the mutations of APP, presenilin (PS), APOE and tau protein genes ,42,96. M1-42 form or modification of the Aβ1-42/Aβ1-40 ratio, depending on the type of mutation [1-42 form in vitro, but do not promote Aβ plaques aggregation in vivo in mice. Several double-transgenic (2xTg) mouse models have been developed by crossing APP and PS1 transgenic mice, demonstrating accelerated Aβ accumulation leading to the impairment of cognitive functions [APP mutations causing cognitive deficits in mice and rats are related to an increased Aβ production, increased Aβ accumulation , promotion of fibrillogenic and toxic Aβmutation ,99,100. unctions ,100,101.In the human tau protein (h-tau) transgenic model, the mice tau gene is replaced by the human gene correlating with NFTs development and neurodegeneration ,102,103.A triple-transgenic mice model of AD (3xTg-AD) is a combination of three mutations resulting in the age-related progressive neuropathy including accumulation of Aβ plaques and NFTs, resulting in the cognitive deficits ,106,107,The number of rat genetic models is much lower . In the TgF344-AD rat model (human APPswe and human PS1ΔE9 mutations) accumulation of Aβ plaques, presence of NFTs, gliosis, increased tau protein level and decreased basal hippocampal synaptic transmission was observed ,110. Aβ Caenorhabditis elegans), zebrafish (Danio rerio) and fruit fly (Drosophila melanogaster) and have been used to create transgenic models of AD [Moreover, several non-mammalian species, including nematode  [i.p.) administration of scopolamine [i.c.v.) injections or infusions of Aβ [i.c.v. injections of streptozotocin (STZ) [s.c.) injections of d-galactose [i.p. injections of MK-801 (a non-competitive NMDA receptor antagonist) [i.c.v. injections of lipopolysaccharide (LPS) [Several pharmacological models of sporadic AD have been used. Those models included: administration of aluminum chloride per os (p.o.) , intrapepolamine ,115, intns of Aβ ,117, i.cin (STZ) ,118, subalactose ,119, i.pagonist) , olfactoagonist)  and i.c.de (LPS) ,123,124 de (LPS) . A modelde (LPS) ,126. It de (LPS) ,125,126.To summarize, it should be pointed out that sophisticated transgenic experimental models of AD are used in preclinical in vitro and in vivo studies on novel drugs or compounds potentially useful in the prevention or treatment of AD. However, those models reflect only genetic changes present in familial AD, whereas the sporadic form of AD is much more common. On the other hand, mostly rather simplistic models of sporadic AD are currently being used in the experimental studies. Some of them are controversial, for example the scopolamine model ,127. It The effects of caffeine have been studied in various transgenic models of AD, including APPswe, double transgenic (2xTg) APPswe/PS1, THY-Tau22 and 3xTg models  104,105,135,136.The most commonly used transgenic models of AD in animals is the APPswe mouse model. In the APPswe model, mice develop cognitive deficits, learning impairment and age-dependent spatial memory dysfunction ,130,131.1R and A2AR hippocampal density was observed in APPswe transgenic mice treated with caffeine [In studies on the APPswe transgenic mice model, caffeine intake prevented cognitive impairment, memory deficits and exerted neuroprotective activity ,131,133.caffeine . CaffeinChronic intake of crude (containing 95.95% caffeine) and pure caffeine prevented memory disorders in APPswe mice model. However, only crude caffeine reduced the level of Aβ and formation of Aβ plaques in the hippocampus. Crude caffeine increased the level of ATP , reduced caspase-3 activity and reduced Aβ-induced neuronal cell death . Crude aStudies on caffeine were also performed on 2xTg and 3xTg animal models. Double transgenic mice indicate rapid rate and early onset of AD associated with cognitive impairment . For exa1-40 and Aβ1-42 levels in plasma (the same effects were observed in transgenic APPswe mice). Similarly, long-term caffeine administration to APPswe/PS1 transgenic mice was related to a permanent and sustained decrease in the Aβ1-40 and Aβ1-42 levels in plasma and reduced Aβ accumulation in the brain. Chronic caffeine administration enhanced cognitive performance in the transgenic mice [Acute intake of caffeine by APPswe/PS1 transgenic mice resulted in a reduced Aβ production (by inhibiting γ-secretase and BACE-1) and reduced Aβnic mice .Acute caffeine use (administered as a coffee constituent) increased the levels of granulocyte-colony stimulating factor—G-CSF, IL-6 and IL-10 in plasma in APPswe/PS1 double transgenic mice and in non-transgenic mice. Higher levels of plasma caffeine were related to lower levels of plasma Aβ in APPswe/PS1 transgenic mice. Chronic intake of concentrated coffee improved cognitive performance and increased the level of G-CSF in plasma in APPswe transgenic mice. The authors concluded that coffee (as a source of caffeine) may protect against AD and that elevated G-CSF levels may contribute to favorable effects of coffee .Also chronic treatment with caffeine in double transgenic APPswe/PS1 mice reversed memory impairment . Caffein2AR improved memory, prevented spatial memory deficits and hippocampal long-term depression in THY-Tau22 transgenic mice [2AR in THY-Tau22 transgenic mice [In a transgenic model of tau disorders, THY-Tau22 mice, progressively developing memory dysfunction is associated with hippocampal neuroinflammation that promotes tau protein hyperphosphorylation and aggregation ,105,139.nic mice . It may Moreover, caffeine exerted anti-inflammatory activity  and antioxidant activity (decreased activity of manganese superoxide dismutase—MnSOD and excitatory amino acid transporter 3—EAAT3 involved in the glutathione synthesis) in THY-Tau22 transgenic mice . HoweverEffects of caffeine were also studied in 3xTg-AD mice . The effects of long-term intake of caffeine in 3xTg-AD mice were assessed in multiple behavioral tests (lasting 21 days) to evaluate sensorimotor functions, exploratory activity, bizarre movements, emotional and anxiety-like behaviors, risk assessment, visual perceptual learning and reference spatial learning and memory in comparison with normal non-transgenic mice. Some of the tests were interpreted as tests concerning BPSD-like behaviors . The effi.c.v. administration of Aβ25-35, in which cognitive dysfunction is observed (Y-maze test and inhibitory avoidance task), acute caffeine administration at a higher i.p. dose or combined 12-day administration in drinking water, followed by acute administration at a lower i.p. dose, prevented Aβ-induced disorders in mice. There was no beneficial effect of the 12-day treatment with caffeine alone [2AR rather than A1R, because similar effects were observed after a selective A2AR antagonist—SCH58261 administration [2AR antagonists also prevented Aβ-induced progressive cognitive impairment and synaptic deterioration in rats [Several non-transgenic models of AD have been used to evaluate the effects of caffeine . In a mone alone . It was stration . In othe in rats ,140.d-galactose to rats is associated with increased oxidative stress resulting in memory impairment, neuroinflammation and neurodegeneration. Concurrent caffeine intake improved memory functions and attenuated cognitive decline in the d-galactose-treated aging rats as demonstrated in the Y-maze behavioral test. Caffeine administration exerted anti-inflammatory activity , TNF-α and IL-1β), antioxidant activity (decreased level of oxidative stress marker—8-oxoguanine) and antiapoptotic activity (decreased B-cell lymphoma 2-associated X protein/B-cell lymphoma protein-2 (BAX/Bcl-2) ratio and reduced caspase-3 and caspase-9 expression) in rats with accelerated aging induced by d-galactose administration. Caffeine treatment alleviated synaptic dysfunction (increasing the level of presynaptic proteins synaptophysin and post-synaptic protein PSD95 in the hippocampus) in d-galactose-treated rats [Long-term administration of ted rats .i.c.v. administration increase oxidative stress, AChE activity and neuroinflammation in the hippocampus and cortex [2AR in the hippocampus (not affecting A1R density) in rats [i.p. STZ injections prevented learning and memory deficits [The STZ-induced AD model is associated with impaired brain glucose metabolism, Aβ accumulation and tau hyperphosphorylation. STZ d cortex ,116,144. in rats . Moreovedeficits ,146.3-induced AD model, administration of aluminum chloride increases level of Aβ, BACE-1, hyperphosphorylated tau protein, proinflammatory cytokines  and oxidative stress markers in the hippocampus and cortex [3 treated rats. Moreover, increased proliferation marker protein—Ki-67 immunoreactivity, decreased glial fibrillary acidic protein (GFAP) immunoreactivity, and increased expression of BDNF and TrkB were observed after caffeine intake [3-induced lipid peroxidation . Caffeine decreased activity of Na+/K+-ATPase in the cerebral cortex, hippocampus and striatum, and reduced activity of AChE in the cerebral cortex and hippocampus. Also anti-inflammatory properties of caffeine were demonstrated (decreased TNF-α level in the hippocampus and striatum) in AlCl3-induced neurotoxicity in rats [In the AlCld cortex ,147. Chre intake . Further in rats .LPS administration induces chronic neuroinflammation and amyloidosis leading to cognitive deficits and memory impairment, reflecting processes specific for sporadic type of AD . Increas1R and A2AR [1R and A2AR antagonists prevented scopolamine-induced memory impairment [Intraperitoneal administration of scopolamine, a non-selective, competitive muscarinic receptor antagonist, is associated with acute memory and attention deficits through cholinergic system blockade ,115. Lonpairment ,152,153.2O2 production, and increased reduced/oxidized glutathione (GSH/GSSG) ratio) and restored A1R level, reduced by cholesterol. There was no caffeine effect on the cholesterol concentration in plasma in rabbits [A high cholesterol diet-induced rabbit model of AD is characterized by an elevated Aβ level in the brain, increased tau protein hyperphosphorylation and disturbed BBB integrity (associated with a disrupted brain cholesterol homeostasis) leading to the learning impairment . Chronic rabbits .Caenorhabditis elegans [Caenorhabditis elegans with human neurotoxic proteins  expression are used to investigate the cellular and molecular mechanisms of neurodegenerative diseases [1-42 in muscles causes, among other things, development of paralysis, whereas its expression in neurons results in their neurodegeneration, and odor preference learning disruption [Caenorhabditis elegans, only products of α-secretase or γ-secretase cleavage (but not β-secretase) are detected, and APP-induced cholinergic neurodegeneration is observed [Caenorhabditis elegans leads to tau aggregate formation and accumulation, neuronal degeneration and synaptic abnormalities resulting in locomotion defects and behavioral impairment [Nematodes are suitable for experimental studies on AD due to their short lifespan, short generation time, transparent body and simple creation of new transgenic lines. Many human genes associated with AD have orthologues in nematodes  elegans ,157. Vardiseases ,159,160.sruption ,159. Aftpairment ,159.Ilex paraguariensis leaves [Paullinia cupana (guarana) seeds [Coffea beans [Caenorhabditis elegans, delaying the paralysis progression in worms and extending their lifespan [v/v, containing 3.6 mM caffeine) treatment did not reduce Aβ aggregation and Aβ distribution. The authors concluded that beneficial effect of coffee probably resulted from skn-1/Nrf2 signaling pathway activation; similar effects were observed after treatment with decaffeinated coffee (0.032 mM caffeine) [Caenorhabditis elegans, and could be mediated by a heat shock factor 1 (HSF-1) and skn-1 [skn-1 and DAF-16 activation [In studies conducted on the nematode AD models, plant extracts containing caffeine were investigated, including the extracts from s leaves , Paullina) seeds , Coffea ea beans , and Zijea beans   . A mixtutivation .Ilex paraguariensis hydroalcoholic extract (IPHE) containing lower concentrations of caffeine (41 or 87 μM). IPHE and caffeine reduced Aβ mRNA levels, decreased AChE activity, increased expression of hsp-16.2 (chaperone protein which overexpression causes suppression of Aβ-toxicity), but also activated DAF-16 signaling . It may suggest that beneficial neuroprotective effects may result from different than caffeine constituents of IPHE [Caenorhabditis elegans.Caffeine alone was investigated only in one study on nematodes . The eff of IPHE . Decaffe of IPHE  and guar of IPHE  also indIn conclusion, the studies on nematode models of AD did not allow the effects of caffeine to be distinguished from those of other constituents of plant extracts ,160,161.2+ in the in vitro culture of the cortical neurons of 3xTg-AD mice  expression in cells exposed to both AlCl3 and Aβ25–35. The effects of caffeine were similar to those induced by selective A1R or A2AR antagonists, indicating the role of blockade of both A1R and A2AR. Caffeine reduced also oxidative stress  activity and decreasing MDA concentration) induced by AlCl3 and Aβ25–35. The authors proposed that combined involvement of A1R and A2AR blockade by caffeine in the neuronal cell protection is associated with the restoration of Ca2+ homeostasis [1R and A2AR, direct or indirect blockade of NMDA receptors and activation of RyRs [Caffeine administered to the culture medium caused an enhanced release of stored Ca-AD mice . The eff the RyR . Similar the RyR . Benefic the RyR ,165. Cafeostasis . In anotViral delivery of mutated human APP and tau protein to primary rat hippocampal neurons and rat dorsal root ganglion (DRG) caused accelerated neuronal cell death and morphological damage (more severe effects were observed in tau transduced cultures). Also, calcium homeostasis dysregulation was observed in APP-EGFP and tau-DsRed2 transduced hippocampal neurons. Moreover, a neurite impairment was demonstrated in APP-EGFP and tau-DsRed2 transduced DRG neurons. Caffeine treatment prevented morphological neuronal damage increasing the number of healthy neurons in APP-induced and tau-induced models .16-22 peptides; the effect was greater when caffeine concentration in the water solution was higher [In silico studies indicated that caffeine may be useful as an anti-amyloidogenic agent in the prevention of AD . Caffeins higher . In silis higher . An imprs higher .So far no drugs have been proven to be effective in reversing or stopping AD. Despite numerous studies having been conducted in recent years, no new drugs have been registered by the FDA for almost 20 years . Due to p.o. or i.p. The descriptions of the doses  used by authors in their reports on in vitro and in vivo studies are not unequivocal. Caffeine consumption in humans differs in a wide range. A low caffeine intake is considered to be below 200 mg/day (<2.86 mg/kg/day), moderate between 200 and 400 mg/day (2.86–5.71 mg/kg/day) and high above 400 mg/day (>5.71 mg/kg/day) [To investigate the effects of caffeine in AD, numerous studies have been undertaken on different experimental models. In those studies, caffeine was administered in different doses. Caffeine was administered in drinking water, in the diet, or once/twice daily /kg/day) . Based o/kg/day) , the cor/kg/day) ,173,174.Generally, experimental studies on caffeine effects in AD demonstrated some beneficial influence on cognition. Its neuroprotective, antioxidant, anti-inflammatory and antiapoptotic activities in the neuronal tissues, led to alleviation of cognitive impairment ,165,166.1R and A2AR), this problem was undertaken in many studies. In fact, both adenosine receptors (A1R and A2AR) are present in synapses of glutamatergic, GABAergic, cholinergic, dopaminergic, serotoninergic and noradrenergic system, and adenosine is involved in neuromodulation of the central nervous system (CNS) [Since the main mechanism of caffeine action is associated with a non-selective blockade of adenosine receptors (mainly Aem (CNS) . Adenosiem (CNS) .2AR (but probably not A1R) in mice with AD induced by Aβ25–35i.c.v. administration [1R may be involved in the protective caffeine activity in cholesterol-enriched diet-induced AD in rabbits [1R and A2AR was responsible for beneficial activity of caffeine in in vitro model of combined neurotoxicity induced by AlCl3 and Aβ25–35 in human neuroblastoma SH-SY5Y cells [In fact, the favorable effects of caffeine were shown to result from the blockade of Astration , in Aβ-istration  and in Sstration . However rabbits . Moreove5Y cells  and in s5Y cells . Dual bl5Y cells .1R and A2AR) have been confirmed by studies with selective A1R and A2AR antagonists. The studies with use of selective A2AR antagonists (MSX-3 and SCH58261) demonstrated that A2AR blockade prevented spatial memory deficits and development of amyloid burden in the double transgenic APPswe/PS1ΔE9 mice [2AR antagonist administration (SCH58261) were observed also in 3xTg  mice [2AR prevented memory impairment and reduced tau protein hyperphosphorylation in THY-Tau22 transgenic mice [2AR antagonist exerts beneficial effects only in slowly progressing memory impairment related to synaptic deterioration [2AR (by a selective antagonist—SCH58261), and also A1R (by a selective antagonist—DPCPX) led to beneficial effects in a scopolamine-induced model in mice [Beneficial effects of caffeine resulting from the blockade of adenosine receptors (AΔE9 mice  and imprΔE9 mice . Similar1L) mice  and in A1L) mice ,140. Delnic mice . Similarnic mice . Howeverioration . However in mice , confirm in mice , but inc in mice .1R and A2AR density was observed in APPswe mice [1AR in rat brains excluding this mechanism of action as the reason for the neuroprotective effect of caffeine in rats [1R and at higher non-toxic doses (<100 mg/kg) A2AR [It should be pointed out that the effects of caffeine on adenosine transmission were not consistent in all studied models. For example, no influence of caffeine on Aswe mice . Moreove in rats . It shoukg) A2AR .2+ release from intracellular stores in the cortical cultures from 3xTg-AD mice [Although numerous studies indicate that antagonistic effect of caffeine on adenosine receptors may be responsible for alleviation of memory deficits, it is not the only possible mechanism of beneficial caffeine action in AD. From among other caffeine mechanisms, the inhibitory effect on PDEs and agonistic effect on RyRs were investigated in in vitro models of AD disorders. Caffeine inhibited PDE, restoring the mitochondrial functions in vitro in mouse N2a-APPswe cells . Activat-AD mice  and in t-AD mice . However-AD mice ,23,25,261-42 deposition, and reduced BACE-1 activity in human neuroblastoma SH-SY5Y cells [3 and Aβ [Numerous mechanisms of caffeine action, which may be relevant to its favorable effects in AD models were demonstrated in studies on cell cultures. For example, in vitro caffeine decreased Aβ level and Aβ5Y cells , and inh5Y cells . Also de3 and Aβ . In addi3 and Aβ . Moreove3 and Aβ . Brewed 3 and Aβ . The eff3-induced neurotoxicity [Since AChE inhibitors  currently play the main role in the treatment of AD, the effects of caffeine on AChE were also studied. In vivo studies demonstrated weak anticholinesterase activity of caffeine in normal rats , in ratstoxicity  and in atoxicity . In vitrtoxicity . This maThe mechanism of favorable actions of caffeine on AD may be connected with the reduction of neuroinflammation ,186. AD d-galactose-treated rats [3-induced neurotoxicity in rats [d-galactose-treated rats [Neuroprotective, anti-inflammatory and antiapoptotic activity of caffeine demonstrated in different experimental models of AD in vivo was reflected in in vitro studies. Caffeine exerted antiapoptotic activity in APPswe transgenic mice ,131 and ted rats . Anti-inted rats , in AlCl in rats  and in tted rats .The caffeine effects presented in studies performed in experimental models of AD are presented in It must be stated that caffeine shares antioxidant and anti-inflammatory effects, leading to neuroprotection, with numerous phenolic compounds, many of them occurring together with caffeine in its dietary sources . In fact, crude caffeine (containing 1% of phenolic acids) exerted stronger favorable effects than pure caffeine . Also, oIt should be pointed out that caffeine did not exert beneficial effects in all experiments on AD experimental models. Caffeine administration did not favorably affect behavioral disorders studied in the behavioral tests in 3xTg-AD mice. Intensification of BPSD-like behaviors and anxiety-related behaviors was demonstrated after caffeine treatment in those mice . The advAs was mentioned before, caffeine  is the most widely used psychostimulant ,192,193.Experimental studies, reviewed in the present article, indicate rather favorable effects of caffeine in animal models of AD. However, results of such studies may not be fully relevant to AD in humans. The AD models have been used to investigate many potential drugs that have proven to be effective in the animals, but this has not necessarily been confirmed in the human studies. Multiple potential disease-modifying drug candidates  failed in the further phases of the trials ,198,199.The studies on transgenic models were limited to the mouse models. The rat studies, carried out on non-transgenic models, were less numerous. In many of the experiments caffeine was administered in drinking water or in the diet, the effect of caffeine used in bolus doses at regular time intervals, over a certain period of time may be different.Another limitation of the experimental studies is that most of them were carried out on male animals , whereas human studies indicate that the effect of caffeine on cognition is sex-dependent ,200,201.In conclusion, the studies carried out on experimental models generally support the notion that dietary caffeine consumption may exert some beneficial effects in AD. However, further studies are necessary to elucidate the role of caffeine in the effects of its sources on cognition and possibly AD risk.


We used pulsed arterial spin labeling (PASL) to investigate differences in cerebral blood flow (CBF) between 26 patients with amnestic mild cognitive impairment (aMCI) and 27 controls with normal cognition (NC). Hypoperfusion was observed in the right temporal pole of the middle temporal gyrus and the right inferior temporal gyrus in the aMCI compared with NC group. Interestingly, hyperperfusion was observed in the left temporal pole of the middle temporal gyrus, left superior temporal gyrus, bilateral precuneus, postcentral gyrus, right inferior parietal lobule, and right angular gyrus in the aMCI group, which likely resulted from a compensatory mechanism to maintain advanced neural activities. We found that mean CBF in the right inferior temporal gyrus, precuneus, and postcentral gyrus was positively correlated with cognitive ability in the aMCI but not NC group. Collectively, our data indicate that PASL is a useful noninvasive technique for monitoring changes in CBF and predicting cognitive decline in aMCI. Mild cognitive impairment (MCI) is an intermediate stage between normal aging and dementia. Amnestic MCI (aMCI) is characterized by memory impairment and is considered to be a prodromal stage of Alzheimer’s disease (AD) . ApproxiArterial spin labeling (ASL) perfusion magnetic resonance imaging (MRI) is widely used to study neurodegenerative diseases . ASL utiHere, we investigated whether there were differences in CBF between subjects with aMCI and those with normal cognition (NC) using pulsed ASL (PASL). Additionally, we evaluated the relationship between altered CBF and global cognition. Our data indicate PASL can detect early stage cognitive impairment and may be useful for monitoring changes in CBF and predicting cognitive decline in aMCI.APOE-ε4 genotype status were observed between the aMCI and NC groups . However, a significant difference in the Mini-Mental State Examination (MMSE) score was observed between the two groups (P = 0.016).Patient demographic and neuropsychological data are shown in Two clusters of hypoperfusion and three clusters of hyperperfusion were observed in the aMCI compared to control group . Hypoperfusion was observed in the right temporal pole of the middle temporal gyrus and the right inferior temporal gyrus in the aMCI group. Additionally, hyperperfusion in the bilateral precuneus , left temporal pole of the middle temporal gyrus , and right inferior parietal lobule (extending to the right angular gyrus) was observed in the aMCI group .APOE-e4 genotype status. A positive correlation between mean CBF in the right inferior temporal gyrus, precuneus, and postcentral gyrus (Clusters 2 and 3) and the MMSE score was observed in the aMCI but not NC group (P < 0.01). No correlation between mean CBF and the MMSE score was observed in the other three clusters  60–85 years of age; 2) intact visual, auditory, and speaking abilities, and 3) willingness to undergo neuropsychological tests and MRI. The exclusion criteria were: 1) history (past year) of the following diseases: local brain injury, traumatic brain injury with loss of consciousness, or immediate confusion caused by traumatic brain injury; 2) anxiety, depression, schizophrenia, or mental retardation; 3) alcohol or drug abuse (past year); 4) severely impaired heart, liver, kidney, or lung function; blood disorders; endocrine diseases; neurosyphilis; 5) history of cancer; 6) ≥ 1 lacunar infarction and patchy or diffuse leukoaraiosis as determined by MRI.The demographic characteristics of the patients including gender, age, and educational background were collected through an interviewer-administered questionnaire. Past medical history including diagnosis with hypertension, diabetes, stroke, or coronary artery disease was obtained from medical records. The following neuropsychological tests were performed: 1) MMSE, 2) Conflicting Instructions Task (Go/No Go Task), 3) Stick Test, 4) Modified Common Objects Sorting Test, 5) Auditory Verbal Learning Test, 6) Modified Fuld Object Memory Evaluation, 7) Trail-making test A&B, and 8) Chinese yuan  Test, which was translated from the EURO Test. Patients with ≥ 6 years of education received tests 1–5 and 7, while subjects with < 6 years of education received tests 1–4, 6, and 8. Normative data and test details are reported elsewhere .All patients received complete medical and neurological exams by professional neurologists. The Center for Epidemiologic Studies Depression and Scale (CESD) guidelines were used to determine whether subjects met the criteria of having a major depressive episode (CESD ≥ 16) within the past week. The Clinical Dementia Rating (CDR) and the Lawton and Brody Activity of Daily Living (ADL) scales were used to assess cognitive complaints, physical self-maintenance ability, and activities of daily living .The consensus diagnosis of MCI or NC was made by an expert panel consisting of two neurologists, one neuropsychologist, and one neuro-epidemiologist based on medical, neuropsychological, and neurological data, and MRI findings. AMCI was defined according to the following criteria : 1) cognAPOE genotyping was performed using the TaqMan SNP genotyping assays. APOE-ε4 positivity was defined as the presence of one or two ε4 alleles [DNA was extracted from blood or saliva samples.  alleles .All subjects underwent MRI at Tongji Hospital using a 3.0-T Siemens Verio MR scanner equipped with a 32-channel head coil. Subjects were instructed to stay relaxed, close their eyes, and keep their head still during the scan. Rubber earplugs were used to reduce noise to a minimum and foam pads were placed around the heads to reduce movement.PASL images were collected using an echo-planar imaging (EPI) sequence with the following parameters: repetition time (TR) = 2,564 ms, echo time (TE) = 11 ms, inversion time (TI) = 700 ms, delay time = 1,800 ms, labeling time = 700 ms, band width = 2,232 Hz / pixel, flip angle (FA) = 90°, thickness = 1.0 mm, field of view (FOV) = 220 mm × 240 mm, matrix = 256 × 256. The overall scan time was 268 s.Whole brain high-resolution anatomical images were acquired using a 3D magnetization-prepared rapid gradient echo (MPRAGE) T1-weighted sequence with the following parameters: sagittal orientation, TR = 21 ms, TE = 3.6 ms, band width = 186 Hz / pixel, FA = 18°, slice thickness = 0.5 mm, slice = 160, FOV = 200 mm × 180 mm, matrix = 384 × 364. The overall scan time was 265 s.http://www.fil.ion.ucl.ac.uk/spm) and ASL toolbox  [The PASL data was processed using the SPM8 software (~zewang) . The cenhttp://restfmri.net/forum/RESTplusV1.2) was considered significant. Age and gender were included as covariates in the regression. CBF clusters were visualized using the xjWiew  toolbox.Whole brain CBF in the aMCI and NC groups was compared using two-sample t-tests. P < 0.05 .Demographic and clinical characteristics were analyzed as follows: continuous variables were expressed as the mean and standard deviation (SD), and were compared using two-sample two-tailed t-tests, while categorical variables were expressed as frequencies (%) and compared between groups using chi-square tests. A generalized linear model (GLM) was used to evaluate the association between mean CBF and MMSE score for each cluster in the aMCI group and NC groups after adjusting for gender, age, education, hypertension, diabetes, stroke, heart disease, and


Regulation of protein expression is essential for maintaining normal cell function. Proteasomes play important roles in protein degradation and dysregulation of proteasomes is implicated in neurodegenerative disorders. In this study, using a proteasome inhibitor MG132, we showed that proteasome inhibition reduces neural stem cell (NSC) proliferation and is toxic to NSCs. Interestingly, MG132 treatment increased the percentage of neurons in both proliferation and differentiation culture conditions of NSCs. Proteasome inhibition reduced B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein ratio. In addition, MG132 treatment induced cAMP response element-binding protein phosphorylation and increased the expression of brain-derived neurotrophic factor transcripts and proteins. These data suggest that proteasome function is important for NSC survival and differentiation. Moreover, although MG132 is toxic to NSCs, it may increase neurogenesis. Therefore, by modifying MG132 chemical structure and developing none toxic proteasome inhibitors, neurogenic chemicals can be developed to control NSC cell fate. Neural stem cells (NSCs) possess the capacity to self-renew and can differentiate into neurons, astrocytes, and oligodendrocytes ,2,3. NSCDifferent types of cells have cell-type-specific protein profile expression; moreover, gene transcription, translation, and protein degradation regulate the distinct proteome expression ,8,9,10. Dysregulation of proteasomes is implicated in various diseases, including cancers and neurodegenerative disorders . It was l-leucyl-l-leucyl-leucine) that has been extensively studied . The membranes were blocked in 5% skim milk or bovine serum albumin  in 20 mM Tris-buffered saline containing 0.03−0.1% Tween 20  for 1 h and incubated overnight at 4 °C with primary antibodies: anti-glial fibrillary acidic protein (GFAP) , TuJ1 , phospho-cAMP response element-binding protein (pCREB) , brain-derived neurotrophic factor (BDNF) , B-cell lymphoma 2 (Bcl-2) , Bcl-2 associated X protein (Bax) , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) , followed by horseradish peroxidase-conjugated secondary antibodies: anti-rabbit IgG  or anti-mouse IgG . Protein bands were detected on a CP-BU New X-ray film  using Western Blotting Luminol Reagent .NSCs were washed with phosphate-buffered saline (PBS) and lysed in a buffer . Cell lysates were centrifuged for 20 min at 25,200× Bdnf, gcccaacgaagaaaaccataag (forward) and gtttgcggcatccaggtaatt (reverse); Bcl-2, tgacttctctcgtcgctacc (forward) and gaactcaaagaaggccacaa (reverse); Bax, tggttgcccttttctactttg (forward) and gaagtaggaaaggaggccatc (reverse); and Gapdh, agttcaacggcacagtcaag (forward) and gtggtgaagacgccagtaga (reverse). The qRT-PCR conditions were as follows: initial activation at 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 15 s, and extension at 72 °C for 20 s. The housekeeping gene Gapdh was used as an internal control.The total RNA was isolated from cells with TRIzol reagent . First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a QuantiTect Reverse Transcription Kit . qRT-PCR was performed with iQ™ SYBR Green supermix . The following primer sets were used to amplify cDNA: ICC analysis was performed as previously described ,39,40. Ct-test .Values are expressed as means ± standard error of mean or mean ± standard deviation, and statistical significance was determined using Student’s To explore the role of protein degradation in the control of NSC fate, we treated NSCs with a proteasome inhibitor, MG132, for 48 h in the presence of mitogens, EGF, and FGF2. NSCs are known to proliferate in the presence of growth factors and differentiate into neurons or glia in the absence of growth factors ,40,41,42To examine the effects of MG132 during NSC differentiation, we treated NSCs with MG132 for 48 h in the absence of growth factors. As shown in To investigate whether MG132 reduces NSC proliferation or MG132 is toxic to NSCs, we measured the size of neurospheres in the medium that promotes proliferation and contains EGF and FGF2 A,B. PhasKi67 is found during all active stages of the cell cycle and is used as a proliferation marker . MG132 sBcl-2/Bax  increases the percentage of neurons although MG132 is toxic, we performed western blotting experiments to determine whether CREB is activated in NSCs upon MG132 treatment. When NSCs were cultured in the presence  and abseControlling protein synthesis and regulating protein degradation are important processes in the maintenance of cell survival and function. Recent evidence suggests that stem cell function and biology can be modulated by proteasomes ,44,45. ARecent data suggest that not only neurons but also astrocytes are essential players for neurodegenerative diseases ,49. AstrAstrocytes can be generated by cytokines including CNTF by activation of signal transducer and activator of transcription 3 and Sma- and Mad-related proteins . TherapeBdnf transcripts. Because MG132 inhibits protein degradation, we expected it to upregulate BDNF protein level; thus, the observed activation of Bdnf gene transcription was somewhat surprising. As MG132 was toxic to NSCs, the cells might have increased BDNF expression as a compensatory protective mechanism. As a result, neurons were the least affected cells when NSCs were treated with MG132 and induced to differentiate. Similar to our results, it has been reported that pluripotent stem cells are extraordinarily sensitive to MG132, whereas motor neurons were relatively resilient to MG132 [It has been known that treatment with MG132 inhibits proteasomes and can mimic the aged status of cells . Our stuto MG132 . Furtherto MG132 . MG132 ito MG132 . In the to MG132 . Recentlto MG132 . Therefoto MG132 ,63. Usinto MG132 ,63,64. Ito MG132 ,66,67. IThe proteasome inhibitor MG132 reduced NSC proliferation and was toxic to NSCs . However


Vitamin D was discovered 100 years ago and since then multiple studies have consistently proved its effect on bone health and mineral metabolism. Further research has also explored its so-called “non-classical” biological effects, encompassing immune regulation and control of cell proliferation and differentiation. Vitamin D downregulates pro-inflammatory immune cells and subsequently their cytokine production, while enhancing the anti-inflammatory subsets, thus mediating inflammation and fostering a more tolerogenic environment. Its biological action is exerted through the vitamin D receptor, a nuclear receptor that mediates gene transcription and is expressed in most cells from the innate and adaptive immunity. Owing to its immune-modulatory properties, its role in cancer pathophysiology, hematology disorders and stem cell transplantation has also been investigated. Vitamin D deficiency causes immune imbalance and cytokine dysregulation, contributing to some autoimmune diseases. In the hematopoietic stem cell transplant setting this could lead to complications such as acute and chronic graft-versus-host disease, ultimately impacting transplant outcomes. Other factors have also been linked to this, including specific polymorphisms of the vitamin D receptor in both stem cell donors and recipients. Nevertheless, studies thus far have shown conflicting results and the use of vitamin D or its receptor as biomarkers has not been validated yet, therefore there are no evidence-based consensus guidelines to guide clinicians in their day-to-day practice. To gain more insight in this topic, we have reviewed the existent literature and gathered the current evidence. This is an overview of the role of serum vitamin D and its receptor as biomarkers for clinical outcomes in patients undergoing hematopoietic stem cell transplantation. Further prospective studies with larger cohorts are warranted to validate the viability of using serum vitamin D, and its receptor, as biomarkers in potential stem cell donors and patients, to identify those at risk of post-transplant complications and enable early therapeutic interventions. Vitamin D has received considerable attention in recent years due to its non-skeletal functions , 2, partantirachitic accessory factor,” “antirachitic vitamin,” or “fat-soluble vitamin”   , 22. In mal fat) .Professor Mellanby extrapolated his research to humans, where lower-social-class children with a diet rich in milk (included those who were breastfed), eggs, or fish had a lower incidence of rickets, better jaws and teeth compared to those from the high class, whose diets were lacking in these aliments .2 and vitamin D3 are absorbed in the small bowels similarly to lipids and then transported to the liver through the lymphatic vessels (Vitamin D is a fat-soluble secosteroid (steroid with a “broken” ring) , 24 main vessels .pro-vitamin D) is transformed into pre-vitamin D3  bounds to vitamin D3 and releases it into the bloodstream (When the solar ultraviolet light B radiation (spectrum 280–320 UVB) hits the epidermis, the 7-dehydrocholesterol   2D3 is the biologically active hormone (24-hydroxylase (CYP24A1) catabolizes 1,25(OH)2D3 into calcitroic acid, functionally inactive. This is excreted through the bile and subsequently the faeces, as well as the urine, avoiding toxic levels  . The quarostatus . This isrostatus . The secCYP27B1) . Calcitr hormone . CYP27B1 hormone , 34. Finc levels . This re . The quc levels , 24. Intc levels .Vitamin D acts as a ligand-inducible transcription factor binding to the VDR, a member of the nuclear hormone receptors superfamily. It is located in most of the cells in humans, including those within the immune system .2D3 itself, including those encoding CYP24A1, leading to an increase catabolism of 1,25(OH)2D3, or CAMP, that enhances the production of cathelicidin, an antibacterial peptide. On the other hand, it downregulates genes, such as those of IL-2 and IFN-γ (interferon gamma) in T cells (granulocyte colony stimulating factor) administration , which is subsequently bound to the Vitamin-D-Responsive Elements (specific sequences of DNA in the promoter region of the vitamin D responsive genes), controlling the transcription of these genes , 37. On stration .The biological functions of vitamin D are divided into classical , 39, 40 VDR is found in cells from innate , 41–46 a3 into its active form because they express the enzyme CYP27B1 2D3 has a very short half life (Immune cells can transform 25(OH)D CYP27B1 , 51, 52. CYP27B1 , 33. Howalf life .Vitamin D targets antigen-presenting cells as follows:in vivo study showed that 1,25(OH)2D3 acts as a differentiation agent in leukemic retinoic acid-resistant promyelocytes into mature granulocytes titution .2D3 increases throughout the maturation of dendritic cells (DCs) due to a higher expression of CYP27B1  CYP27B1 . Howeverolerance , 55, 56. surface , 55–57,  surface , 44. It 2D3 synthesis. In addition, vitamin D regulates the expression of specific endogenous antimicrobial peptides, such as cathelicidin  proliferation and cytotoxic function is abrogated by 1,25(OH)nd IFN-γ , 61. In nd IFN-γ .2D3 into calcitroic acid and subsequently to eliminate it 2D3 downregulates the production of IL-2 and IFN-γ by Th1   in the T cell surface, with a subsequent impairment in T cell trafficking  , 66. Theing IL-2 . Alongsifficking , 68. Thifficking , 70, 72.Moreover, a preclinical study postulated that a population of IL-10-secretor B cells could act as regulatory immune cells, but data is limited so further research is needed .Currently, vitamin D deficiency is considered a pandemic disease . AlthougMany factors have been identified to contribute to it: age , low sun3 has been estimated to be between 2 and 3 weeks. It identifies individual adequacy or insufficiency, making it the most useful marker of the vitamin D body stores Dy stores .3 required to prevent bone loose while maintaining calcium homeostasis  (NICE guidelines and the Endocrine Society Task Force on Vitamin D established it below 25 nmol/L (10 ng/mL)  , NICE gu0 ng/mL) , 85, and0 ng/mL) . TherefoMoreover, the non-skeletal functions of vitamin D have reinvigorated its interest as potential modulator in a broad spectrum of diseases and therapeutical procedures, as follows:Despite some clinical studies focused on the role of vitamin D deficiency have revealed its contribution to the pathophysiology of some autoimmune diseases, including multiple sclerosis, systemic sclerosis, rheumatoid arthritis, insulin-dependent diabetes and systemic lupus erythematous , 45, 87,+ T cells, leading to a poor recruitment of Tregs. However, it can be restored with vitamin D supplementation: Due to its immunomodulatory properties, vitamin D enhances the secretion of IL-10 by CD4+ T cells, increasing the pool of both population of circulating Tregs (Foxp3+ and IL-10 Tregs) in vitro and contributing to the control of the disease, as seen in clinical studies Dt sepsis . Howeverrculosis  or downrrculosis , data rerculosis , 92, 93.rculosis .In vitro studies have shown that 1,25(OH)2D3 inhibits cellular proliferation (downregulating BCL-2 expression and telomerase activity) and angiogenesis , and acts as a pro-apoptotic and differentiation-inducing agent in a range of malignant cells D3 had a higher neutrophil count at the time of engraftment raftment . Neverthraftment , 82. Morraftment , 117, bu2D3 stimulates proliferation and differentiation of CD34+ hematopoietic stem cells em cells –120. It em cells –123. Howem cells .GvHD is a major complication following allogeneic HSCT and one of its main causes of death . ClinicaSurprisingly, vitamin A has also been suggested to be involved in GvHD pathogenesis , but its2D3 serostatus and acute GvHD (aGvHD): Urbain et al. demonstrated that patients with moderate to severe aGvHD had lower levels of 25(OH)D3 after HSCT ter HSCT . Kreutz ter HSCT . Finallyter HSCT . Neverthter HSCT , 84, 1153 prior to HSCT had a higher cumulative incidence of Chronic GvHD (cGvHD) and extensive cGvHD compared to those with higher levels Dr levels . Supportantation . In contantation , 132.Currently there is controversy in the evidence of the impact of vitamin D deficiency within the GvHD pathophysiology. Therefore, further studies with larger sample size to confirm this are warranted.More than 50% of patients treated with steroids for GvHD are resistant to this immunosuppressive treatment –135. The3 were associated with poorer steroid response Dresponse , 137. Neseverity , 72, 90.steroids , 72, 138In the steroid-resistant GvHD setting, one preclinical study suggested that synergism between vitamin D supplementation and steroids could abrogate the monocyte-induced release of pro-inflammatory cytokines and therefore mitigate the tissue damage by GvHD .These findings serve as a rationale for treating or preventing vitamin D deficiency by upholding normal levels of vitamin D in order to enhance the immunosuppressive effect. Since vitamin D may overcome the resistance to immunosuppression in GvHD, further research in this field is needed to confirm this hypothesis and potentially to reduce the morbidity and mortality associated to this disease.As previously described, vitamin D has an immune-modulatory role, and it may protect against infections and blunt tissue damage on the course of HSCT , 91. OwiFokl FF and ApaI aa reflect upregulation of the VDR activity, whereas ApaI AA downregulates it, impacting on the activity of Th1 and Th2 on the early immune reconstitution following HSCT D0 nmol/L . Awarene0 nmol/L , 151–1550 nmol/L .3 prior and after HSCT, and commence on replacement therapy if clinically indicated. Nevertheless, no conclusions were reached regarding the ideal threshold for vitamin D deficiency due to the lack of robust studies including HSCT patients Dpatients . DiffereVitamin D is a potent regulator of immune responses with impact in HSCT –13. NeveThe high incidence of vitamin D deficiency in allogeneic HSCT patients, alongside the current controversy , 141, emVitamin D deficiency may be the first potential easily modifiable host factor associated with post-allogeneic HSCT outcomes, thus identifying patients at high risk and optimizing its management to enable prompt therapeutic intervention is encouraged.JSo lead the manuscript writing and CA, AM, and JSn contributed to it. All authors accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.