Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53’s effects on stemness could lead to novel therapeutic strategies in cancer research.Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53’s functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 ( In fact, in leukemia cells expression of wtp53 led to apoptosis confirming the tumor suppressor function of wtp53 [wtp53, are bypassed by damaged cells when p53 is inactivated or mutated leading to uncontrolled growth and carcinogenesis [Our knowledge of p53 has developed extensively over the past four decades as scientists have unraveled its distinct biological processes. p53 has been associated with several functions in normal cells and cancer. The discoveries by six independent laboratories in 1979 have become the focal point igniting what is now decades of research devoted to p53. These studies found the 53-kDa protein to be bound to Large Tumor Antigen in Simian Virus SV40) infected tumor cells. In the same period, a separate group identified p53 in transformed but not normal mouse cells from reactions with mouse sarcoma antiserum ,3,4,5,6.0 infecteof wtp53 ,15,16,17ogenesis ,19.Most of the investigated functions of p53 are associated with full-length p53 (TAp53). Twelve isoforms are encoded in the TP53 gene: TAp53  ∆40p53 , ∆133p53  and ∆160p53  ,21,22,2315, Ser20, Ser33, Ser37, Ser46, Thr18 and Th81, which block the Mdm2 binding to p53. The C-terminus of p53 can be phosphorylated to activate p53 DNA binding activity at Ser315 and Ser392. Acetylation at Lys320, Lys373 and Lys382 or SUMOylation at Lys386 has also been observed in this region [In response to these stress factors, p53 is stabilized through posttranslational modifications via phosphorylation, acetylation, ubiquitination, methylation, neddylation, or SUMOylation ,27,28,29s region ,31. Onces region ,35. Genos region ,25,36.In another example of p53 regulation through a negative feedback loop, ubiquitin ligases Cop1 and Pirh2 associate with p53 before undergoing ubiquitination and proteosomal degradation ,38. The wtp53 loss of functions as a tumor suppressor. Similarly, tumor-associated mutp53 acquires gain-of-function (GOF) oncogenic properties that are independent of wtp53. These mutations enable cancer-associated mutp53 with new properties that benefit the tumor at various levels of cancer progression. Mutp53 can exert its GOF properties through binding and inactivation of p53 associated proteins such as p53 family members p63 and p73. Mutp53 GOF processes are associated with a variety of physiological events: tumor cell proliferation, invasion, survival, metabolic changes, angiogenesis, metastasis, tissue modeling, enhanced chemoresistance, mitogenic defects, genomic instability and stem cell expansion [Mutp53 has been shown to upregulate numerous genes, including MDR1, PCNA, EGFR, IGF1R, c-myc and IGF2 [Mutp53 can also downregulate pro-apoptotic genes such as caspase-3, and CD95/Fas/Apo1, and wtp53 target genes including p21 and PTEN [p53 mutations generated by missense mutations in the TP53 gene have been implicated in tumor progression as a result of xpansion ,49,50,51xpansion . Mutp53 and IGF2 ,56,57,58and PTEN ,60,61,62The p53 family includes p63 and p73, which share similar gene structures and overlapping, but distinct functions cover a wide range of processes involved in development, apoptosis and cell cycle arrest, chemosensitivity and immortalization . p63 isoStem cells are a rare population of cells with the innate capacity to self-renew and give rise to a lineage of progenitor cells that can mature and propagate into a variety of cell types. Residing in specific tissues, stem cells provide functions critical to the needs of their location while mediating homeostasis, maintenance and repair ,70,71,72−/−) mice, an increase in the detection of tumorigenic melanoma cells by several orders of magnitude was observed. Limiting dilution assays using unselected patient primary and metastatic melanoma cells from 12 patients revealed that 25% resulted in tumor formation, while tumors formed from 27% using unselected cells from four patients. Kelly et al. (2007) [CSCs or tumor-initiating cells (TICs), are a rare population of cells with the ability to initiate the growth and maintenance of a heterogeneous tumor. They have been identified in a variety of hematopoietic cancers, solid tumors and tissues, including those of the bladder, breast, brain, lung, cervix and prostate ,75,76,77. (2007)  suggesteThe origins of CSCs have not been completely revealed. However, it has been proposed that CSCs are long-lived adult stem cells or progenitors with accumulated neoplastic transformations that have enabled them to sustain cancer growth, progression and metastasis ,87,88. IBesides tumor initiation and cancer relapse, CSCs have been purported to be a cause of metastasis. Due to the variety of CSC properties, subpopulations have been proposed to include primary CSCs (pCSCs) that induce tumor formation and metastatic CSC (mCSCs) that induce metastases . The frewtp53 can inhibit CSCs. p53 recruits histone deacetylase 1 (HDAC1) to the CD133 promoter and reduces histone H3 acetylation. Reduction in CD133 expression inhibits tumor cell proliferation, colony formation and the expression of Nanog, Oct4, Sox2 and c-Myc stemness transcription factors [Stem cells and cancer cells have a highly proliferative potential giving rise to normal and malignant heterogeneous tissues with cells exhibiting unique clonogenic properties ,102,103. factors .Transcription factors serve critical functions in ESC self-renewal, and many are overexpressed in tumors. Somatic cell reprogramming into iPSCs can be initiated by epigenetic changes and the expression of transcription factors Sox-2, c-Myc, Oct-4, Klf-4 and Lin-28 . Also asmutp53 plays a role in this population as wtp53 is important to non-malignant stem cells. CSCs are less susceptible to chemotherapy compared to the vast majority of the tumors due to a slow proliferation rate, efficient DNA repair, high expression of anti-apoptotic proteins and ABC multi-drug pumps [Since CSCs behave similarly to non-malignant stem cells, it is not surprising that ug pumps ,112,113.ug pumps ,114.Stem cell processes are controlled by intrinsic factors that are cell-autonomous transcription factors and genes and a network of extrinsic factors that include soluble factors and adhesion molecules from ECM and niche cells . Stem cemutp53, through prolonging of NF-κB activation and cell survival, can promote the inflammatory environment needed for the development of colorectal tumors. Ubertini et al. (2015) [mutp53 GOF in promoting inflammatory signals by the repression of the secreted interleukin-1 receptor antagonist (sIL-1Ra). The findings demonstrate that mutp53 tumorigenesis requires sIL-1Ra suppression, resulting in a chronic pro-inflammatory tumor microenvironment. mutp53 influences tumor and microenvironment interactions and may induce pro-oncogenic GOF changes in tumors and stromal cells alike. As demonstrated by Addadi et al. (2010) [mutp53-expressing fibroblasts could promote tumor growth better than p53 null fibroblasts.The tissue microenvironment is a powerful line of defense against tumorigenesis. Embryonic skin in the uterus can inhibit tumor formation of implanted murine B16 malignant melanoma cells. Likewise, chick and zebrafish embryonic microenvironments have been observed to inhibit tumor formation of implanted cancer cells ,131,132.. (2015)  reported. (2010) , mutp53-Mutp53 GOF activities have been identified in cell reprogramming, expansion, maintenance and interaction with the tumor stroma. It is well known that wtp53 is a suppressor of somatic stem cell reprogramming, preventing differentiated somatic cells from being reprogrammed into pluripotent stem cells with unlimited expansion ability. Mutp53 can initiate tumor formation by promoting the generation and expansion of pluripotent cells. For example, loss of p53 results in the de-differentiation of somatic cells, while mutp53 enhances reprogramming [mutp53 R238Q transgenic mice exhibited an expansion in HSC and mesenchymal stem cell (MSC) progenitors. Similar to cancer cells, CSCs may modulate the microenvironment through cell-to-cell contact or the secretion of factors. Topczewska et al. (2006) [gramming ,138,139.gramming  reported. (2006)  showed t−/− astrocytes are de-differentiated into CSC-like cells while maintaining neural stem and progenitor phenotypes and self-renewing activities, undergo neurosphere formation in vitro and form tumors in vivo [Recent studies have implicated p53 in stem cell plasticity in the areas of self-renewal, differentiation and reprogramming and have associated these processes with tumor cell heterogeneity and progression. For example, focal adhesion kinase (FAK), p53 and Nanog have been found to be interlinked and to cross-regulate CSCs. FAK binds to Mdm2 and activates p53 degradation, whereas p53 upregulation inhibits Nanog and FAK survival signaling pathways, increasing CSC maintenance, cell growth and tumor inhibition . Loss of in vivo .mutp53 GOF processes occur through multiple pathways. Fiore et al. (2014) demonstrated that mutp53 GOF causes differentiation of human osteosarcoma MG-63 cells into 3AB-OS CSCs. They investigated the expression of wtp53 in 3AB-OS CSCs compared with parental MG-63 cells. Parental cells contain a single copy of wtp53 gene, and the promoter is methylated. 3AB-OS cells contain a rearranged TP53 gene of multiple copies by the P72R polymorphism and hot spot mutation R248W. Ectopic expression of p53-R248W/P72R lends to a cancer a stem-like phenotype, possessing a high proliferative rate with clonogenic growth, undergoes sphere formation and exhibits strong features of invasiveness and migration. Stem cells and p53 have been associated with Wnt/β catenin signaling pathways, Sonic Hedgehog (Shh), BMI-1 (B-cell-specific Moloney murine leukemia virus insertion site 1), Notch and PTEN pathways that provide a delicate balance of self-renewal and differentiation through niche signaling and the interplay among these pathways [Increasing evidence suggests that these diverse pathways ,126,145.wtp53 and mutp53 in non-malignant somatic stem cells and CSCs. Recognizing the similarities between ontogeny and oncology, p53 deficiency and stemness may collectively contribute to tumor initiation and recurrence through survival advantages, such as: disruptions in cell death programming, drug resistance and self-renewal. In non-malignant cells, it has been shown that p53 can influence stem cell homeostasis and pluripotency. wtp53 has been observed to counteract somatic cell reprogramming and constrain iPSC generation in vivo [mutp53 acquires GOF processes to influence stemness as observed in its ability to stimulate iPSC formation while augmenting the malignant potential of reprogrammed cells [Mutp53 GOF induces EMT transition by modulating the miR-130b-ZEB axis in endometrial cancer [Accumulating evidence demonstrates that p53 loss can lead to an acquisition of stemness in cancer, highlighting the role of  in vivo ,149,150. in vivo . p53 may in vivo . In tumoed cells . p53 caned cells ,155,156.ed cells . Mutp53 l cancer . These el cancer  to propo−/− iPSCs exposed to DNA damage, carry short telomeres and chromosome aberrations and are genetically unstable [Like p53 mutants, p53 isoforms have also been implicated in promoting CSC potential. Overexpression of the p53 isoform p53∆133p53β promotes stemness in MCF-7 breast CSCs by regulating the expression of stem cell factors Sox2, Oct3/4, Nanog and CD24/CD44 and promoting mammosphere formation  showed tunstable . By decrunstable . p21, a unstable  showed twtp53, mutp53 and p53 isomers to stemness in both non-malignant stem cells, such as ESCs, iPSCs and tumor CSCs. It is becoming increasingly evident that p53 may possess a stemness role in non-malignant and CSCs beyond its main tumor suppressor function. p53 suppresses self-renewal and induces differentiation in embryonic development and organogenesis. In response to DNA damage, mouse ESCs (mESCs) activate p53 to bind to the promoters of self-renewal and pluripotency genes Nanog and Oct4, evoking differentiation into cell states responsive to p53-induced cell cycle arrest or apoptosis [wtp53 and inhibition of mutp53 and CSC-associated p53 isomers might lead to the reduction of drug resistance and enhance treatment efficacy  [mutp53 facilitated somatic cell reprogramming and augmented the malignant potential of reprogrammed cells. Likewise, Fiore et al. (2014) [mutp53-R248W/P72R exhibited GOF activity in 3AB-OS MG63 osteosarcoma cells with its ectopic expression promoting aggressiveness, chemoresistance and cancer stemness. While investigating tumor progression in epithelia ovarian cancer, Ren et al. (2016) [mutp53 regulation of tumor differentiation and responsiveness to steroids. Furthermore, Lu et al. (2013) [mutp53 R175H GOF in programming mammary tumorigenesis by expanding mammary epithelial stem cells (MESCs) that generate tumors. However, the molecular mechanisms for how p53 promotes stemness and whether the stem-like cells acquire survival advantages for tumor initiation and progression are not clearly understood [In cancer, loss of p53 tumor suppressor function often accompanies cleation ,173,174.. 2010)  reported10  repor. (2014)  found th. (2016)  reported. (2013)  demonstr+/ABCG2+ hESCs through transient suppression of p53 activity. The enhanced stemness was transient; however, it provided a highly cytoprotective state with enhanced transcriptional activity of Oct-4 and Nanog in undifferentiated cells. The hESCs also demonstrated high Bcl-2, fibroblast growth factor 2 (FGF2), altering the oscillation system state of p53/Mdm2 [Oxidative stress activates p53 during tissue injury hypoxia resulting in death and differentiation of hESCs. However, hESCs are able to avoid this fate by exerting a cytoprotective effect. Das et al. (2012) reported conditional reprogramming activity or “enhanced stemness” by elevating HIF2α transcriptional activity in SSEA3p53/Mdm2 . Itahanap53/Mdm2  showed ivimentin induction, cytoskeletal changes, cell adhesion and ECM degradation [+/CD24−/low), enabling tumorsphere formation [mutp53 background promoting the induction of SLUG expression [EMT is a reversible process in which polarized epithelial cells lose their cell-cell adhesions and acquire a spindle-like morphology. The accompanying mesenchymal properties enable cells to migrate and invade distant tissues. This process is essential for gastrulation, neural crest delamination of vertebrates, tissue formation, embryonic cell development, wound healing, fibrosis, carcinogenesis and metastasis ,182,183.radation ,184,185.radation ,187. In ormation ,186,188.ormation . During ormation ,190. Cicormation  demonstrormation ,193,194.ormation ,175,195.pression ,197.wtp53 directly activates transcription of miR-200 and miR-34 gene loci, creating reciprocal feedback loops to reduce EMT inducers ZEB1 and SNAIL [wtp53 directly activates the miR-200c and miR-192 transcription leading to ZEB1/2 downregulation and EMT repression [Mutp53 promotes EMT and tumor aggressive potential by inhibiting wtp53-miR-200c pathways via dominant-negative effects on wtp53 [mutp53 GOF facilitates EMT and cancer cell invasion through repression of the ZEB1 inhibitor, miR-130b. Shi et al. (2014) [Chang et al. 2011)  showed tnd SNAIL ,199,200.nd SNAIL ,201,202.1  showednd SNAIL . Korpal nd SNAIL  reportedon wtp53 . Dong eton wtp53  demonstr. (2014)  reportedwtp53, participate in cell cycle progression, angiogenesis, programmed cell death, viral duplication and cell differentiation [wtp53 triggers senescence in human tumor cells lacking functional p53. Dividing and proliferating cells frequently experience more DNA double-strand break damage than do non-proliferating cells [−/− hESCs have been used to show that the role of p53 in suppressing pluripotency is conserved in mouse and human ESCs. In MEFs, Ferbeyre et al. (2002) reported that oncogenic ras and p53 cooperate to induce senescence through activation of the MAP kinase pathway [In response to genotoxic stress, the tumor suppressor p53 induces transient growth arrest or apoptosis and mediates a cellular process of limited proliferation and irreversible growth referred to as senescence and the cell cycle G1/S checkpoint. p53 isoforms have also been implicated in replicative senescence and, similar to ntiation ,209,210.ntiation . Sugrue ntiation  demonstrng cells ,214. mESng cells . p53−/−  pathway . Gannon  pathway  showed tContemporary chemotherapies can obliterate the majority of dividing and proliferative cancer cells in the tumor; however, the inability to completely eradicate CSCs ensures tumor recurrence . CSCs haIn hair follicle stem cells (HFSCs), the state of quiescence is a form of tumor suppression. Cancer cells originating from HFSCs give rise to cutaneous squamous cell carcinoma. Tumorigenesis is averted when the cell cycle is stalled at G0/G1, suggesting that the processes maintaining HFSCs’ dormancy are dominant over oncogene gain  or p53 tumor suppressor loss. Many intrinsic mechanisms known to regulate quiescence include transcription factors FoxO, HIF-1α and NFATc1, and signaling through ATM and mTOR. Multiple extrinsic regulatory mechanisms in the microenvironment have been identified, including bone morphogenic protein (BMP), osteopontin, thrombopoietin (TPO), angiopoietin-1 (Ang-1), tumor growth factor-β (TGF-β), N-cadherin and integrins, as well as Wnt/β-catenin signaling . PTEN iswtp53 and p53 null mice revealed Gfi-1 and Necdin to be p53 target genes. Gfi-1 maintains HSC functional integrity and inhibits HSC proliferation, while Necdin is a negative cell cycle regulator increasing quiescence.p53 is essential for restraining cell cycle entry. Many studies have associated p53 with regulation of stem cell quiescence. Loss of p53 in neural stem cells (NSCs) and HSCs triggers stem cell expansion, as these cells exit quiescence and progress through the cell cycle ,228. FurItahana et al. (2002)  reportedwtp53 function is necessary for probing the mechanisms of p53 inactivation in CSC genesis and tumor development. Loss of wtp53 function is prevalent in more than half of all solid and hematologic malignancies and persists as mutations or deletions altering p53 activity levels. Wtp53 found in cancers is nonfunctional in that p53 is either degraded by Mdm2, or secluded from the nucleus inhibiting its function in transcription [wtp53 loss of function [Molecule development to explore novel targets and signaling pathways to restore cription ,233,234.function .mutp53 in CSCs and improve therapeutic strategies, including chemotoxic agents , mutp53 reactivators  or wtp53 inhibitors  [Combinatorial therapies have been employed to target c agents . Many of, PFT-µ) ,248,249.mutp53 has also been employed [MET, and modifies thiol groups in the central domain of mutp53 to restore tumor suppressor p53 [mutp53 in CSC from breast, endometrial and pancreatic cancer cell lines, allowing for the reduction in tumor cell growth and inhibition of tumorsphere formation. In solid tumors, small molecule MIRA-1 can induce apoptosis through p53-dependent processes [Huang et al. (2009) used Ellipticine with 5-FU as a strategy to deplete deficient p53 DLD1 colon CSC. The use of small molecules, such as CP-31398, MIRA1 or PRIMA-1, to induce transactivation activities in employed ,251,252.employed ,243. PRIssor p53 . Zhang essor p53  used PRIrocesses .mutp53 to wtp53, including the transcriptional activity of hotspot p53 mutants p53R273H and p53R175H, such as peptide CDB3 with l-amino acid sequence REDEDEIEW [mutp53 and restore wtp53 function.Synthetic peptides derived from known p53 binding proteins can restore the conformation of EDEDEIEW . PeptideEDEDEIEW ,257,258.EDEDEIEW . PositivEDEDEIEW . These pEDEDEIEW . BispeciEDEDEIEW  demonstrRecent studies have discovered RING finger E3 ligases to be important contributors to cell stemness via regulation of self-renewal, differentiation and drug resistance displayed by CSCs. Inhibition of RING finger proteins may provide promising strategies for cancer treatment. RING finger proteins are not catalysts with active sites; instead, they facilitate ubiquitin transfer from an E2 directly to the substrate. Disrupting the RING structure or RING-E2 interface would represent viable routes for inhibitor development. Nutlin-3 is a small molecule antagonist, which binds to the amino terminus of Mdm2 and prevents binding to p53, leading to increased stability of p53 . As a reAs the guardian of the genome, p53 maintains cellular integrity by inhibiting the cell cycle and preventing the proliferation of damaged cells . These pwtp53 [ESCs and early embryos endogenously express high levels of full-length p53 and remain viable upon removal of p53 ,266,267.wtp53 . The MDMResearchers have embarked on associations of Mdm2 with stemness in a variety of cellular processes. Daniele et al.  (2015) d6-methylguanine DNA methyltransferase (MGMT). Removal of the methyl group from TMZ induces chemoresistance in glioma stem cells and is prevalent in a significant fraction of glioblastoma cases. Sato et al. (2011) [In patients with newly-diagnosed glioblastoma, temozolomide (TMZ) is the standard of treatment; however, a challenge exists in overcoming TMZ drug resistance mainly due to O. (2011)  show tha−/− p53 515C (encoding p53R172P) mice were generated resulting in normal HSC counts in fetal livers and depleted counts in postnatal bone marrow. These mice are also observed with increased ROS levels and p53R171P expression, activation of p16 lnk4a and cell death. The experimental study signifies the importance of the p53-Mdm2 pathway in hematopoiesis. In the model, basal ROS levels accumulate in hematopoietic tissues during development. The absence of Mdm2 in combination with basal oxidative stress permits p53 activity to induce ROS production via transactivation of PIGs, creating a positive feedback loop. HSCs and progenitors become depleted via the induction of cell cycle arrest, senescence and cell death. Sensitization of bone marrow HSCs to the ROS-p53 pathway can be exploited in leukemia, because leukemia stem cells can propagate from HSCs. During normal hematopoiesis, stress signals activate p53, and Mdm2 is required for p53 level regulation to enable stem cell survival [Additionally, Abbas et al. (2010)  reportedsurvival .Stem cells serve an indispensable role in normal development and organ regeneration. Stem cells and progenitors can differentiate into various tissues in the body, repair damaged tissue and serve as a resident population of quiescent cells throughout the life of an organism. However, these cells may undergo neoplastic transformation, leading to dysregulation in normal stem cell processes. Uncontrolled self-renewal and differentiation result in deleterious consequences on development, aging and tumorigenesis. p53 is involved in several distinct processes that influence normal stem cell functions, maintaining stem cell number and quality.Functioning as a guardian between non-malignant stem cells and CSCs, p53 prevents stem cell dedifferentiation and propagation of cells with genomic abnormalities. Many advances have been made in unraveling the mechanisms of cancer progression. The tumor suppressor p53 has been an investigative focal point for nearly four decades. Despite the advances in current cancer therapeutics, advanced tumors continue to evade our most versatile treatments due to the inability to effectively target and eliminate CSCs. It is now evident that a link has been revealed between the concept of “stemness” and its regulation by the guardian of the genome, p53, in non-malignant stem cells and CSCs.wtp53, p53 isomers, mutp53 and p53 binding and regulatory factors, such as Mdm2, have been implicated in the stemness regulation processes. Targeting these CSC pathways and their intricate and interactive tumor milieu may prevent tumor recurrence following surgery, chemotherapy and radiation.In order to adequately treat cancer and ensure the success of President Barack Obama and Vice President Joe Biden’s 2016 National Cancer Moonshot Initiative, it is imperative that future studies focus on understanding the mechanisms that maintain stable genomes in stem cells and how these signaling pathways are deregulated in the genesis of CSCs and CSC-mediated tumor recurrence. Studies have revealed similar signaling pathways among non-malignant stem cells and CSCs. The Wnt, Hedgehog, BMI-1, Notch and PTEN evolutionarily-conserved pathways have demonstrated regulatory roles in the self-renewal and the cell fate decision of embryonic and hematopoietic stem and progenitor cell, as well as the maintenance of CSCs. mutp53 constitutes a multitude of proteins. Most investigations of mutp53 have been explored at mutations in the amino acids 175, 245, 248, 249, 273 and 282 in the DNA binding domain. Likewise, other domains have been found to be mutated, but their contribution to tumorigenesis and stem cell regulation have not been thoroughly investigated [mutp53 protein function in a variety of oncogenic and stem-conferring processes. The frequency of missense mutations differs in subclasses of tumors of the same organ. This is evident in breast cancers where luminal types mostly exhibit p53 point mutations, whereas basal tumors mostly detect alternations resulting in p53 truncation [mutp53 upstream and downstream stem pathways and interacting partners, such as PTEN and MDM2.p53 isomers encompass at least 12 different p53 proteins, and stigated . Differeuncation . UnderstMdm2 gene. How are Mdm2 protein functions dependent on activated signaling pathways and the many Mdm2 isoforms generated from alternative start sites or mRNA splicing? How does Mdm2 switch from ubiquitinating to a neddylating E3 in CSCs? Thus, it is imperative to completely understand how Mdm2 is regulated and how it regulates stemness as targeting Mdm2 to prevent CSCs may inadvertently promote CSC stemness and tumor heterogeneity. Thus, the complexity of p53 gene splicing and mutations and its regulatory network is currently evolving in the context of CSC, and further studies are necessary to gain critical information on how CSC may or may not respond to p53-targeted therapy.When investigating the processes contributing to stemness, it is vital to understand which factors come into play. The frequencies of CSCs were once believed to be as rare as 0.0001% to 0.1% of all cancer cells ,85. Rece


Recurrence and metastasis are the usual manifestations of treatment failure of epithelial ovarian carcinoma (EOC). Vasculogenic mimicry , aldehyde dehydrogenase 1 , KiSS-1 (suppressor of tumor metastasis), and metastasis associated in colon cancer-1 (MACC1) are all useful predictive factors for metastasis and prognosis in various cancers. In this study, we analyzed associations among VM, ALDH1, KiSS-1, and MACC1 in EOC, and their respective correlations with clinicopathological characteristics and survival in EOC.Positive rates of VM, ALDH1, KiSS-1, and MACC1 in 207 whole EOC tissue samples were detected by immunohistochemistry. Patients’ clinical data were also collected.Levels of VM, ALDH1, and MACC1 were significantly higher, and levels of KiSS-1 significantly lower, in EOC tissues than in benign ovary tumors. Levels of VM, ALDH1, KiSS-1, and MACC1 were associated significantly with tumor/lymph node/metastasis (LNM) grade, implantation, and International Federation of Gynecology and Obstetrics (FIGO) stage, and with patients’ overall survival (OS); whereas the KiSS-1+ subgroup had significantly longer OS than did the KiSS-1− subgroup. In multivariate analysis, high VM, ALDH1 or MACC1 levels, FIGO stage, implantation and low KiSS-1 levels were independently associated with shorter OS in patients with EOC.VM and expressions of ALDH1, KiSS-1, and MACC1 represent promising markers for metastasis and prognosis, and potential therapeutic targets for EOC. In 2012, epithelial ovarian carcinoma (EOC) was reportedly found in 238,700 newly diagnosed cases of ovarian cancer (OC), and caused 15,1900 deaths . As OC iAlthough vasculogenesis and angiogenesis have been shown to promote tumor growth and metastasis, clinical benefits of anti-angiogenesis therapy for cancers are still unsatisfactory . TreatmeTumor recurrence and metastasis may be related to cancer stem cells (CSCs), also named tumor-initiating cells, which have the ability to self-renew and initiate heterogeneous cancer cells that compose the tumors . They maAldehyde dehydrogenases (ALDHs) are found in the cytoplasm, nucleus, and mitochondria , and affInactivation of metastasis suppressor genes and activation of metastasis-promoting genes are early events in cancer invasiveness and metastasis. KiSS-1, a suppressor of metastasis, was first identified in melanoma, through analysis of subtractive hybridization . It is lMetastasis-associated in colon cancer 1 (MACC1) was the firstly identified by Stein et al. in colon cancer in 2009 . MACC1 iOverall, studies of VM, ALDH1, KiSS-1, and MACC1 in association with tumor metastasis and prognosis suggest that these factors affect cancer progression; however, the associations among VM, ALDH1, KiSS-1, and MACC1 in EOC has not been widely reported. In this study, we examined the hypothesis that these factors are mutual correlated and are related to metastasis and prognosis in EOC.th edition of the International Federation of Gynecology and Obstetrics (FIGO). Tumors were graded according to World Health Organization standards  who were treated for EOC at the First Affiliated Hospital of Bengbu Medical College, from January 2008 to December 2010, along with 60 samples of benign tumors (serous- or mucinous-cystadenoma). Patients who had received preoperative chemo- or radiotherapy, or other anti-cancer therapy, were excluded. All tissue samples  were obtained with patients’ consent. This study was approved by the ethics committee of Bengbu Medical College and performed in accordance with the guidelines of the Declaration of Helsinki (No.BBMCEC2012063).. We included patients for whom we had complete pathological, clinical, and follow-up data . Overall survival (OS) was calculated from the patient’s surgery date to her death date or December 2015 . As 20 patients had lost contact due to relocation, the cohort for survival data was 187 patients. Tumor-node-metastasis (TMN) stage was assessed according to the 2014TM Plus detection kit instructions . All EOC and control tissues were fixed in 10% buffered formalin and embedded in paraffin. Continuous 4-μm-thick sections were cut. All EOC and control sections were deparaffinized and dehydrated in xylene and graded alcohol, then washed with phosphate buffer saline . Endogenous peroxidase activity was blocked by incubating sections in methanol containing 3% H2O2 for 10 min at room temperature (RT), then placed in citrate buffer (pH 6.0) and heated to 95 °C for antigen repair for 30 min. After several washes with PBS, all sections were quenched with goat serum at RT for 30 min, then incubated with mouse monoclonal antibody against human CD34 , ALDH1 (Abcam), KiSS-1 , or rabbit polyclonal antibody against human MACC1 (Santa Cruz Biotechnology) at 37 °C for 1 h. All samples were conducted periodic acid-Schiff (PAS)–CD34 dual staining to determine endothelial cells in glycosylated basement membranes of vessels, as well as vessel-like (VM) structure [Immunohistochemistry was performed according to the Elivisiontructure . FurtherA modified Yue’s method was used to assess VM in the EOC tissues and the control tissues . All speStaining results were assessed semi-quantitatively by two independent pathologists who were blind to patients’ clinical and follow-up data. Ten high-power-fields (HPF) from different areas of each EOC slide were analyzed to avoid any intratumoral heterogeneity of antibody expression. Staining results were scored according to intensity  and extent . Intensity and extent scores were then multiplied to yield final scores that ranged 0–12. Scores ≥3 were considered positive. For sections that were positive for all three of ALDH1, KiSS-1, and MACC1, an average of the final score of each section was taken.P < 0.05 was considered significant.Associations between clinicopathological characteristics and VM, ALDH1, KiSS-1, or MACC1 were compared using Fisher’s exact test or Chi-square test. Associations between VM, or ALDH1, or KiSS-1, or MACC1 was compared using Spearman’s coefficient test. Effects of VM, ALDH1, KiSS-1, or MACC1 on survival were determined by univariate and multivariate analyses. Independent prognostic factors were determined using the Cox regression model for multivariate analysis. The Kaplan–Meier method with log-rank test for univariate OS analysis was used to evaluate associations between VM+, ALDH1+, KiSS-1+, or MACC1+ results and clinicopathological characteristics, using SPSS 19.0 software for Windows . P < 0.001; Fig. To evaluate the contributions of VM, ALDH1, KiSS-1, and MACC1 to EOC, the results thereof were immunohistochemically evaluated for both EOC and control tissue samples. These data were then compared to patients’ clinicopathological characteristics. The rate of VM+ findings  in the EOC samples 36.2%; 75/207) was significantly higher than that in the control tissues  than in control tissues  than in control tissues  than in control tissues , LNM , implantation , and FIGO stage  compared with those with VM− , ALDH1 , or MACC1 . Expression of ALDH1 and that of VM  and MACC1  were positively associated, as were VM and MACC1 , cell motility, invasion, and metastasis , 28, 42.FIGO staging guides therapeutic strategies for patients with EOC, but does not provide clear information about EOC’s behavior. Therefore, novel and effective biomarkers to predict EOC behavior, metastasis, and patient prognosis are urgently needed. In this study, multivariate Cox regression analysis indicated that VM, ALDH1, KiSS-1, and MACC1 expression and implantation, as well as FIGO stage, are independent prognostic factors for EOC patients. Our results thus support VM, ALDH1, KiSS-1, and MACC1 as credible biomarkers for EOC, especially in predicting metastasis and prognosis.Moreover, as ALDH1 is a marker for CSCs, its involvement in CSCs may play an important role in the initiation and progression of EOC. CSCs can induce angiogenesis to provide adequate nutrition and oxygen for rapid tumor growth , and canOur results imply that ALDH1 affects EOC evolution; and that combined detection of VM, ALDH1, KiSS-1, and MACC1 are valuable indicators of metastasis and prognosis in EOC.


The purpose of this study is to identify key genes and microRNAs (miRNAs) involved in autoantibody-mediated arthritis (AMA).R was employed to identify differentially expressed genes (DEGs) in samples collected at days 1–18 compared with those collected at day 0. Consistent DEGs were obtained by taking the interaction of DEGs from different time points, followed by functional enrichment analysis. MiRNAs were screened out and constructed into regulatory network with DEGs using Cytoscape.A time-course microarray data (ID: GSE27492) of peripheral blood leukocytes, ankle tissue, and synovial fluid from K/BxN mouse serum-transferred mice were downloaded from Gene Expression Omnibus. Those samples were collected at days 0, 1, 3, 7, 12, and 18 after serum injection. Limma of Ephx1 and upregulated AF251705, Adam8, Arg1, Basp1, Ccl2, Ccl7, Ccl9, Ccr2, Clec4a2, Clec4d, Cxcl1, Fabp5, Fcgr1, Gp49a, Il1rn, and Saa3. Those DEGs were associated with biological processes of immune response, inflammatory response, and defense response; chemokine signaling pathway; cytokine-cytokine receptor interaction; and NOD-like receptor signaling pathway. Additionally, 202 miRNAs were identified to have a regulatory effect on 9 of the 17 DEGs. Notably, miR-944, miR-374a, and miR374b were found to regulate the expression of Cxcl1, Ccl7, and Ccl2. Clec4d was targeted by 78 miRNAs.In total, 17 consistent DEGs were obtained, including downregulated Our study reveals that 17 DEGs and 202 miRNAs may be associated with autoimmune disorder in the progression of AMA, which could guide future researches. IL-1) [IL-6 [IL-18 [MMP-1 and MMP-13. Those uncovered genes demonstrate an involvement of the gene factor in the progression of arthritis.Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized by inflammation of the joints and surrounding tissues and destruction of bone and cartilage . InflammIL-1) , IL-6 [66 [IL-18 , and CC 6 [IL-18 , especiaIL-1 [Mouse models with autoantibody-mediated arthritis (AMA) are widely used in researches of arthritis . The K/BIL-1 . Monach IL-1 . NeutropIL-1 . FollicuIL-1 . Thus, tMicroRNAs (miRNAs) representing a family of non-coding RNAs are shown to play an essential regulatory effect on the innate immune system in rheumatic diseases . MiRNA-3To systemically investigate the molecular mechanisms underlying AMA, the time-course gene expression profiles deposited by a previous study  were re-Microarray data set GSE27492 deposited by Jacobs et al.  was downR. For the several probes corresponding to a single gene, their expression levels were averaged as the final expression level for the gene. Differential expression analysis was performed with package limma [P value < 0.05 were set as the cut-offs to screen out DEGs. The expression levels of genes from samples collected at day 1, 3, 7, 12, and 18 were separately compared with those from samples collected at day 0. The consistent DEGs were obtained for further analysis by taking the intersection of the DEGs emerging at all of the five time points.Raw data were processed through background correction and median normalization using robust multi-array average method  from pacge limma . |log FChttp://david.abcc.ncifcrf.gov/) [P value < 0.05 was chosen as the threshold to screen out significant GO terms and KEGG pathways.To identify altered biological pathways in arthritis, Gene Ontology (GO) enrichment analysis  and Kyotrf.gov/) . P valueThe miRNA-targeting DEGs were predicted based on 10 databases covering DIANA, MiRanda, MiRDB, MiRwalk, RNAhybrid, TargetScan, RNA22, PITA, PICTAR5, and PICTAR4. The predicted miRNAs from more than 5 of the 10 databases were identified as candidate miRNAs. Following, the regulatory network between miRNAs and DEGs was constructed using Cytoscape software .Ephx1  and upregulated AF251705 (cDNA sequence AF251705), Adam8 , Arg1 , Basp1 , Ccl2 (chemokine (C-C motif) ligand 2), Ccl7 (chemokine (C-C motif) ligand 7), Ccl9 (chemokine (C-C motif) ligand 9), Ccr2 (chemokine (C-C motif) receptor 2), Clec4a2 , Clec4d , Cxcl1 (chemokine (C-X-C motif) ligand 1), Fabp5 , Fcgr1 , Gp49a (glycoprotein 49 A), Il1rn (interleukin 1 receptor antagonist), and Saa3 (serum amyloid A 3) , molecular functions of chemokine activity, chemokine receptor binding, and cytokine activity , as well as cellular components of extracellular space and extracellular region part . KEGG pathway enrichment analysis uncovered the DEGs were associated with chemokine signaling pathway , cytokine-cytokine receptor interaction , and NOD-like receptor signaling pathway .With the cut-off of ed Table . GO enriCxcl1 and Ccl7; miR-374a and miR374b both could regulate the expression of Ccl7 and Ccl2. Additionally, Clec4d was targeted by 78 miRNAs, suggesting an important role in the regulatory network.Using the 10 databases for predicting miRNAs, 202 persuasive miRNAs were identified to have a regulatory effect on 9 of the 17 DEGs, including miR-155, miR-152, miR-124, miR-1, and miR-206 (Fig. Focusing on screening molecular targets for AMA therapy, this study obtained 16 up-regulated and 1 downregulated genes in the mouse with AMA compared with the mouse without AMA by re-analyzing the time-course microarray data. Large troop of miRNAs was also screened to have a regulatory effect on 9 of the 17 DEGs based on the predictive databases. Enrichment analysis unmasked the potential molecular mechanisms by which the DEGs take part in the development of AMA.3C groups and their receptors are revealed to play key roles in leukocyte migration to inflamed synovium in the treatment of chronic inflammatory disorders of RA [Adam8, Saa3, Il1rn, Ccl2, Ccl7, Ccl9, Ccr2, and Cxcl1. GO enrichment analysis revealed that immune response and inflammatory response were significantly enriched biological processes of DEGs, which were linked to RA and in line with previous studies [Cxcl1, Ccl2, Ccr2, Fcgr1, and Ccl7 were also associated with molecular functions of chemokine activity, chemokine receptor binding, and cytokine activity. Thus, a disorder of chemokine-associated functions was also observed in this study. In addition, Adam8 is detected under several pathological conditions and may be a potential drug target for the treatment of inflammatory diseases [Cxcl1, which may be a target of anti-TNF therapy [CCR2 is regarded as a therapeutic target in RA treatment [CCR2 blockade cannot sufficiently induce clinical improvement in RA patients [CCL2 monoclonal antibody treatment also results in poor clinical improvement [Fcgr1 makes a contribution to arthritis pathology [Clec4a2 and Clec4d play essential roles in maintaining the homeostasis of immune system [Arg1 activity is important for wound healing functions and immune response to viral infections [Chemokines consisting of CXC, CC, C, and CXrs of RA . Accordi studies , 27. Thodiseases . IL-33-i therapy . CCR2 isreatment . These fpatients .The antirovement . It coule system , 35; Argfections . TherefoCcr1, Ccr2, and Ccr5 are associated with macrophage and endothelial cell infiltration in arthritis [NOD2 signaling could be used as a therapeutic target for the control of RA [Cxcl1 and Ccl2 by Nod1 stimulation [Correspondingly, KEGG pathway enrichment analysis revealed the DEGs were associated with chemokine signaling pathway, cytokine-cytokine receptor interaction, and NOD-like receptor signaling pathway. Those pathways are closely related to inflammatory and immune responses. The upregulation of chemokine receptors rthritis . The cytrthritis . The nucrthritis . The NODol of RA . Mesothemulation . ConsideCxcl1, Ccl2, and Ccl7. MiR-944 located in the intron of TP63 gene could promote cell proliferation, migration, and invasion in human cervical cancer [Cxcl1, Ccl2, and Ccl7. Additionally, Clec4d was targeted by 78 miRNAs. Previous report has pointed out activated Clec4d can contribute to Mincel expression and thus enhance the immune system [Clec4d may play a role in AMA as a downstream target of various miRNAs. Although the role of the above miRNAs and Clec4d in RA development has not been fully investigated, based on our results, we speculate that these molecules may serve as potential targets for RA treatment. All these speculations need further research. There were some limitations in our study. The comparative studies of other arthritis models were not included this study. Moreover, the results were obtained based on the bioinformatic methods and should be further confirmed by mechanistic experiments. Even so, this study reveals 17 DEGs associated with AMA, which may provide a theoretical basis for further studies.Furthermore, a large cohort of miRNAs was identified to be associated with the DEGs and the miRNAs were constructed into a regulatory network with DEGs. MiR-155, miR-152, and miR-124 have been shown to be implicated in RA –44. MiR-l cancer . MiR-374l cancer . Althouge system . It coulClec4d probably mediates the functions of a large number of miRNAs in the development of AMA. Further experiments, both epidemiological and mechanical, should be performed to confirm the results.Focusing on screening of therapeutic targets for AMA, this study reveals that 17 dysregulated genes may jointly and consistently play roles in the progression of AMA by affecting the immune system functions and pathways. MiR-944, miR-374a, and miR374b may be associated with AMA development by targeting some chemokines. Also,


Enterobacter aerogenes is a motile, non-spore forming, Gram-negative bacteria from the Enterobacteriaceae family. Enterobacter spp. have emerged as multidrug-resistant (MDR) nosocomial bacteria, especially in intensive care units  clone carrying the blaTEM-24 gene  among 16 infected patients . The main selection criterion for genome sequencing was the MDR phenotype, particularly in carbapenem resistant isolates. The negative laboratory tests for carbapenemases were also taken into account, as divergent enzymes or alternative resistance mechanisms could be relevant to the observed MDR phenotypes. C10 and D2 samples were isolated from different body sites of the same patient. Isolates were grown in selective medium with an ertapenem disk (10 ug) and stored at −80°C in trypticase soy broth containing glycerol 15%. Identification of isolates was performed using Vitek® 2 Compact  and by mass spectrometry using Microflex LT instrument . This study was carried out in accordance with the Brazilian legislation and was approved by the Institutional Ethics Review Board of the Hospital de Clínicas, Universidade Federal do Paraná (IRB#: 2656.263/2011-11). Our study involved only bacterial isolates and no human specimens were analyzed or stored. Further, we used no patient information other than the anatomical sites from where the isolates were collected. Therefore, the same Ethics Review Board exempted us from obtaining informed consent forms.Isolates were tested by agar dilution against 15 antibiotics according to the Clinical and Laboratory Standard Institute guidelines CLSI, . MinimalE. aerogenes isolates were determined by pulsed-field gel electrophoresis (PFGE), as described elsewhere . DNA quality was assessed using a Bioanalyzer 2100 system . DNA quantification was performed using Qubit . Illumina sequencing libraries with an average fragment size of 550 bp were prepared using Illumina TruSeq DNA PCR-free LT Kit . Whole-genome sequencing of paired-end (PE) libraries was performed using a HiSeq 2500 instrument in RAPID run mode  at the Life Sciences Core Facilities of the State University of Campinas . Quality-based trimming and filtering was performed using Trimmomatic version 0.32  to detect carbapenemases were positive for C10, D2, and E9. However, no class A, B, and D carbapenemase encoding genes were detected by PCR. All isolates tested negative in carbapenem hydrolysis assays.All isolates showed MDR profile and had increased MIC for at least one carbapenem. Information regarding collection date and site, clinical setting, PFGE profile and antimicrobial resistance profiles of each isolate are available in Table We obtained between 16,841,714 and 25,138,390 150 bp PE reads per library. After genome assembly, 5,833,521 bp were assembled in 58 scaffolds for C10, 5,821,782 bp were assembled in 57 scaffolds for D2, 5,584,745 bp were assembled in 55 scaffolds for D3 and 5,637,471 bp were assembled in 59 scaffolds for E9. By using the NCBI Prokaryotic Annotation Pipeline, we were able to predict 5,363, 5,380, 5,067, and 5,129 protein-coding sequences in each of the genomes listed above, respectively. Genomic features of the four sequenced genomes are summarized in Table aacA4 and aadA); (ii) beta-lactam resistance, including genes belonging to class A beta-lactamases (TEM family), class B beta-lactamases (Ribonuclease Z), class C beta-lactamases (CMY/LAT/MOX/ACT/MIR/FOX family) and class D beta-lactamases (OXA-9); (iii) bacitracin resistance (gene bacA), and (iv) sulphonamide resistance  and overexpression of AcrAB-TolC efflux pumps  of each isolate were deposited in Sequence Read Archive (SRA) under the accession numbers SRP083774 (E. aerogenes C10), SRP083784 (E. aerogenes D2), SRP083785 (E. aerogenes D3), and SRP083786 (E. aerogenes E9). Users can download the data for research purposes, citing the present manuscript as original reference.The genome sequence of AG, NV, JP, LD, and TV conceived the idea and designed the study. JP performed the sample collections and wet lab experiments. AG and NV carried out the genome analysis. AG, NV, JP, LD, and TV interpreted the data and wrote the manuscript. All authors have read and approved the final version of this 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.


Salvia miltiorrhiza is a traditional Chinese medicinal herb used for treating cardiovascular diseases. Depside salt from S. miltiorrhiza (DSSM) contains the following active components: magnesium lithospermate B, lithospermic acid, and rosmarinic acid. This study aimed to reveal the mechanisms of action of DSSM. After searching for DSSM-associated genes in GeneCards, Search Tool for Interacting Chemicals, SuperTarget, PubChem, and Comparative Toxicogenomics Database, they were subjected to enrichment analysis using Multifaceted Analysis Tool for Human Transcriptome. A protein-protein interaction (PPI) network was visualised; module analysis was conducted using the Cytoscape software. Finally, a transcriptional regulatory network was constructed using the TRRUST database and Cytoscape. Seventy-three DSSM-associated genes were identified. JUN, TNF, NFKB1, and FOS were hub nodes in the PPI network. Modules 1 and 2 were identified from the PPI network, with pathway enrichment analysis, showing that the presence of NFKB1 and BCL2 in module 1 was indicative of a particular association with the NF-κB signalling pathway. JUN, TNF, NFKB1, FOS, and BCL2 exhibited notable interactions among themselves in the PPI network. Several regulatory relationships  were also found in the regulatory network. Thus, DSSM exerts effects against cardiovascular diseases by targeting JUN, TNF, NFKB1, FOS, and BCL2. Salvia miltiorrhiza  is widely used as a traditional Chinese medicinal herb to prevent and treat vascular diseases  were associated with all MLB, RA, and LA; four genes  were associated with both MLB and RA; and four genes  were associated with both MLB and LA. Jun protooncogene (JUN), TNF, nuclear factor kappa B subunit 1 (NFKB1), and Fos protooncogene (FOS) were noted as particular hub nodes according to their BC, closeness centrality, and DC scores (κB) signalling pathway  and leishmaniasis (p = 4.72E−07) were particularly associated with the nodes in modules 1 and 2, respectively   and 108 relationship pairs (such asCL2/TNF) . JUN → TNF/FOS, FOS → NFKB1, and NFKB1 → BCL2/TNF) were found to be involved in the transcriptional regulatory network.In this study, a total of 73 DSSM-associated genes were identified from various databases. In the PPI network constructed on the basis of these genes and their interactions, JUN, TNF, NFKB1, and FOS were established as hub nodes according to their BC, closeness centrality, and DC scores. JUN, TNF, NFKB1, FOS, and BCL2 were also shown to interact among themselves in the PPI network. Moreover, two distinct modules (modules 1 and 2) of the PPI network were identified. In addition, several regulatory relationships  across endothelial cells, thus contributing to the reservoir of LDL in vascular walls [ TNF may be associated with the functions of DSSM in cardiovascular diseases.Prolonged anti-TNF-soriasis . Studiesrthritis , 29. TNF setting . TNF-α bar walls . These f (DN-c-Jun) gene transfer inhibits VSMC proliferation, and JUN is associated with the intimal hyperplasia induced by balloon injury [ FOS is a novel factor that determines the severity and development of atherosclerosis and is thus involved in tobacco toxicity in coronary artery disease (CAD) patients [ miR-146a-targeting FOS is a potential tool for treating enhanced inflammation-associated cardiac disorders [ JUN and FOS may also be targets of DSSM in cardiovascular diseases.Blocking the JNK pathway may provide a new strategy for treating the cardiomyocyte death induced by myocardial ischaemia/reperfusion , 33. Domn injury . Saliquepatients . Palomerisorders . Thus, J NFKB1 polymorphism (rs28362491) may be a genetic marker of CAD [κB, which is in turn correlated with the reduction of plasma inflammatory markers [ BCL2 expression, miR-21 promotes heart failure progression with preserved left ventricular ejection fraction and subsequently inhibits cardiac fibrosis [ miR-1 targets BCL2 functions in mediating cardiomyocyte apoptosis [ NFKB1 and BCL2 in module 1 indicated a particular association with the NF-κB signalling pathway, indicating that NFKB1 and BCL2 may be correlated with the effects of DSSM against cardiovascular diseases through this pathway.Studies involving Uygur and Han women in China have demonstrated that the DD genotype ofr of CAD , 38. The markers . By incrfibrosis . Moreovepoptosis . In this JUN, TNF, NFKB1, FOS, and BCL2 were also revealed as potential targets of DSSM for treating cardiovascular diseases. However, further experimental research should be performed to confirm these findings obtained by bioinformatic analysis.In conclusion, a total of 73 DSSM-associated genes were identified by conducting a search of public databases.


The objective of the present study was to investigate differences in the expression of apoptosis-related factors in the eutopic and ectopic endometrium (EuE/EE) in women with adenomyosis before and after laparoscopic bilateral uterine artery occlusion (LUAO). Ten patients with uterine adenomyosis who received LUAO were selected as the research subjects, from whom EuE and EE tissues were obtained before and after LUAO and detected for the expression of apoptosis-related molecules in EuE and EE by PT-PCR and Western blot, and changes in the mitochondrial structure by electron microscopy. Normal endometrial stromal cells (NESC), and EuE/EE stromal cells in women with adenomyosis were cultured in a 1% O2, 5% CO2 incubator to establish a physical anoxia state in an in vitro stromal cell model. The expression of apoptosis-related molecules was observed at 0, 6, 12, 24 and 48h of hypoxic. The results showed that the expression of apoptosis-related factors in EuE and EE were increased significantly after LUAO and under hypoxic conditions in vitro, suggesting that transient ischemia and hypoxia were involved in the apoptosis of adenomysis lesions, and that uterine artery occlusion could remove adenomyosis lesions on tissue/cell level by cytoreduction, thus reaching the goal of treating adenomyosis effectively. Adenomyosis is a common chronic disease primarily diagnosed in childbearing females. It is defined as the presence of endometrial glands and stroma causing reactive hyperplastic or hypertrophic myometrium., surrounded by chronic inflammation in the endometrium ,2,3,4. UBased on a series of LUAO treatments of uterine fibroids, we originally propose the hypothesis of ‘Single organ uterus shock’ in our previous study, which may ideally explain the UAO mechanism in theory . After LAntibodies for Apaf-1, CHOP, TRADD, Bcl-2 and Bax were purchased from Santa . Antibodies for Endo-G, Cyt-c, AIF, GRP78, Caspase3, Caspase4, Caspase8 and Caspase-9 were purchased from Abcam . Antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technologies . Secondary antibodies of goat anti-mouse FITC, goat anti-rabbit HRP and goat anti-mouse HRP were purchased from the Beyotime Institute of Technology.Ten patients with uterine adenomyosis who received LUAO combined with partial resection of adenomyosis were recruited in this study. The preoperative diagnosis of adenomyosis patients based on typical clinical symptoms of dysmenorrhea and/or menorrhagia and pelvic pain, physical examination findings, and imaging results including transvaginal ultrasound and MRI. The ages of the patients ranged from 34 to 45 years with a mean of 39.67±5.01 years. All control women and adenomyotic women had regular menstrual cycles (28–35 days). The exclusion criteria were patients who were diagnosed with uterine fibroid and subsequently received hormonal therapy within the past 6 months; patients with reproduction tract infections or immune system and endocrine diseases; and patients whose preoperative hysteroscopy excluded the diagnosis of endometrial lesions. The phase of the menstrual cycle was the early proliferative phase. Fresh EuE and EE tissues before and after LUAO were collected, and EuE and EE tissues obtained before LUAO were used as control. Part of them were stored at 4°C in phosphate-buffered saline (PBS)  and processed within 2–18 h, and the remaining part were stored at -80°C for RNA and protein extraction. In hypoxia cell models experiment, normal cell control was from patients who voluntarily required to place intrauterine device (IUD) excluding other diseases, without the use of hormone drugs nearly 3 months. Samples of endometrium obtained by pipelle or curettage before place the IUD were cultured. Details of the LUAO procedure and the sampling time were recorded. All cases included in this study were confirmed by postoperative pathology.The study was approved by the Ethics Committee of Yang-Pu Hospital, Tongji University School of Medicine , and in accordance with the tenets and guidelines of the Declaration of Helsinki. All patients provided written informed consent.EuE and EE samples were fixed in glutaric acid and osmic acid, dehydrated with pyruvic acid, epoxy resin embedding, sliced into ultra-thin sections, stained with uranyl and lead citrate, and finally observed under an electron microscope for mitochondrial morphological changes and cell apoptosis such as vague or disappearing mitochondria cristae, mitochondrial swelling or cavity changes.5' TCTACCTCTGCTGACAAG 3', reverse primer: 5' CACCGTTTGAGACATTCC 3'; CHOP, forward primer: 5' CAGGAAACGGAAACAGAG 3', reverse primer: 5' CACCATTCGGTCAATCAG 3'; TRADD, forward primer: 5' CCAGCCCTTACAGTTTCAC 3', reverse primer: 5' GGCAGGCAAGATTGATTCC 3'; Bcl-2, forward primer: 5' AGACCGAAGTCCGCAGAACC 3', reverse primer: 5' GAGACCACACTGCCCTGTTG 3'; Bax, forward primer: 5' AGCTGAGCGAGTGTCTCAAG 3', reverse primer: 5' TGTCCAGCCCATGATGGTTC 3'; Endo-G, forward primer: 5' TGTCTGCACAGGGCCACTCTTC 3', reverse primer: 5' TGCCTCCAGGATCAGCACCTTG 3'; Cyt-c, forward primer: 5' CTTTGTGCTGGAAGGAACC 3', reverse primer: 5' CAGGGCATTACACGCTAAC 3'; AIF, forward primer: 5' GCTACAAGCACGCTCTAAC 3', reverse primer: 5' GCCCAAATCACTCCAGAAC 3'; GRP78, forward primer: 5' CCCGTCCAGAAAGTGTTG 3', reverse primer: 5' CAGCACCATACGCTACAG 3'; Caspase3, forward primer: 5'GTTTGAGCCTGAGCAGAGAC 3', reverse primer: 5' TGGCAGCATCATCCACAC 3'; Caspase4, forward primer: 5' AATGGAGCTGACTTTGAC 3', reverse primer: 5' ATAAAGCAGCACATCTGG 3'; Caspase8, forward primer: 5' CTGGGAGAAGGAAAGTTG 3', reverse primer: 5' TTGGAGAGTCCGAGATTG 3'; Caspase-9, forward primer: 5'GGGTCGCTAATGCTGTTTC 3', reverse primer: 5' TGCTAAGAGCCTGTCTGTC 3'; GAPDH, forward primer: 5' CACCCACTCCTCCACCTTTG 3', reverse primer: 5' CCACCACCCTGTTGCTGTAG 3'. The SYBR Green kit was used, and GADPH served as an internal control. The mRNA expression level of the apoptosis-related genes Apaf-1, CHOP, TRADD, Bcl-2, Bax, Endo-G, Cyt-c, AIF, GRP78, Caspase3, Caspase4, Caspase8 and Caspase-9 in EuE and EE tissues following LUAO was detected by real-time PCR. The 2-ΔΔCT method was used to calculate the mRNA levels [Liquid nitrogen-preserved tissues were homogenized, and total RNA was extracted using the TRIzol method . A reverse transcription kit was used to prepare cDNA. The following PCR primer sets were used in this study: Apaf-1, forward primer: A levels .Western blotting analyses was performed to detect protein expression. Primary antibodies were incubated overnight with the appropriate primary antibody and secondary antibody. Finally, the optical density of protein signal strength was determined, using GAPDH as an internal standard to determine the protein level. The primary antibodies included anti-rabbit Bcl2 , anti-rabbit Bax , anti-rabbit cyt-c , anti-rabbit AIF , anti-rabbit caspase3 , anti-rabbit caspase 4 , anti-rabbit Caspase 8 , anti-rabbit Caspase 9 , anti-rabbit EndoG , anti-rabbit Apf1 , anti-rabbit GRP78 , anti-rabbit CHOP , anti-rabbit TRADD , and anti-rat GAPDH .Three samples of fresh EE in Hank’s Balanced Salt Solution (HBSS) containing double resistance  were obtained and rinsed 2–3 times. Next, 8 ml 0.4% collagenase (containing DNase I: 20 μg/ml) and 2 ml 0.05% pancreatic enzyme were added to the samples and digested at 37°C with shaking for 2–3 hours. Then, the tissues were cultured in DMEM-F12 medium  in a 37°C incubator. According to the adherent time differences of the stromal and epithelial cells, we obtained stromal cells. According to the identification of the cell morphology, stromal cells exhibited flat, spindle-shaped, fibroblastic-like morphology, and interstitial cell surface marker (vimentin) of the separated stromal cells, we established a separate cell culture system. Using this preparation method, three groups  were established. Three p2, 5% CO2 incubator to establish a physical anoxia state, and a normal oxygen (21% O2) condition for 0, 6, 12, 24 and 48h. Next, the cells were collected, and Western blot was performed to identify the protein expression of the main apoptosis-related factors Bcl-2, Bax, caspase 3, Endo-G, caspase 8 and GRP78. The obtained results were compared between the groups.An in vitro stromal cell model was established, and then the experiment cells, including normal endometrial stromal cells (NESC), EuE stromal cells (EuESC) and EE stromal cells (EESC) were cultured in a 1% OUsing GraphPad Prism 4 (GraphPad software), data were expressed as the mean ± SD. Tissue samples were analysed using the paired t-test, and cell specimens were analysed using an independent t -test. P<0.05 was considered statistically significant.Electron microscopy (30000×) revealed that EuE and EE tissue in adenomyosis underwent apoptosis after LUAO. The mitochondria cristae looked vague or disappeared with mitochondria swelling and a cavitation change. In addition, there was a nuclear chromatin edge and cell membrane reflex. The cytoplasmic concentration and the formation of apoptotic bodies were observed .RT-PCR was used to detect the gene expression of Apaf-1, CHOP, TRADD, Bcl-2, Bax, Endo-G, Cyt-c, AIF, GRP78, Caspase3, Caspase4, Caspase8 and Caspase-9 in 10. As shown in Western blot was performed to detect the protein expression of apoptosis-related factors in EuE and EE tissues after LUAO in the 10 specimens. EuE and EE tissues obtained before LUAO were used as the control group, and those obtained after LUAO were used as the experimental group. The protein expression of Bax, AIF, caspase-3, caspase-4, caspase-8, caspase-9, GRP78, Apf-1, CHOP and TRADD was detected by Western blot. .Primary NESCs, EuESCs and EESCs were cultured for in vitro experiments. The detailed procedures for the isolation, identification and cultivation of the NESCs, EuESCs and EESCs were described previously .Western blot was used to detect the apoptosis-related factors. The result showed that the protein expression levels of apoptosis-related molecules  in NESCs, EuESCs, EESCs was increased significantly at 0, 6, 12, 24 and 48h of hypoxia in a time-dependent manner .In the present study, we used LUAO to treat patients with adenomyosis and observed changes of EuE and EE tissues by electron microscopy. The result showed that EuE and EE tissues underwent apoptosis after LUAO, presenting typical apoptosis characteristics such as swollen mitochondria, cavitation sample change, nuclear chromatin edge, cell membrane reflex, cytoplasmic concentration and the formation of apoptotic bodies. Then we found the expression of apoptosis-related factors in EuE and EE were increased significantly after LUAO and under hypoxic conditions in vitro.The main purpose of the current clinical treatment of uterine adenomyosis is to remove the lesions, relieve the symptoms, reduce recurrence, spare the uterus and maintain fertility. By using LUAO combined partial resection, we have achieved good outcome in the clinical treatment of adenomyosis in recent years ,13. Our In the ischemic anoxia-induced intrinsic apoptotic pathway, mitochondria transmit different signals to induce stress, such as molecules from the Bcl-2 and Bax families, to maintain the stability of the membrane. When this balance is impaired, apoptosis-related proteins, such as cyt-c, AIF, and Endo-G, are released from the mitochondria, and cyt-c is transferred into the mitochondrial electron transport chain and released into the cytoplasm where it can bind to Apaf-1 to initiate a downstream caspase cascade, activating apoptosis . In addiWe constructed a cell hypoxia model in vitro to determine the anoxic conditions at 0, 6, 12, 24 and 48h and selected apoptosis-related proteins in the mitochondria and endoplasmic reticulum to confirm the results. In CESC, EuESC and EESC cells, changes in the expression of caspase-3, Endo-G, caspase-8, GRP78 and Bax showed a positive correlation with the duration of anoxia, particularly for caspase-3, Endo-G and caspase-8 in adenomyosis. However, the expression of Bcl-2 was negatively correlated with hypoxia and inhibited expression. Cytological experiments with prolonged hypoxia confirmed the apoptotic changes and showed a significant difference. The cytology experiments also revealed that with prolonged hypoxia, more significant changes in apoptosis were observed, and these changes were more obvious in the adenomyotic EuE and EE than those in the normal endometrium, although the mechanism remains to be further elucidated. This phenomenon may be correlated with our LUAO solution because the main body combined surgical treatment had a good clinical effect in uterine adenomyosis. However, the effect of LUAO joint lesion resection treatment of adenomyosis on ovarian and reproductive function has been debated. In the early stage, we selected LUAO preoperative patients and followed them up postoperatively for 1, 3 and 6 months to assess the impact of FSH, LH, E2, and INHB (serum inhibin B) on ovarian function, and the results showed no significant difference [In summary, Our study confirms that the EE has experienced more significant apoptosis after LUAO, which may clarify the precise mechanisms so as to lay a theoretical basis for the clinical treatment of the adenomyosis. However, there is still a need for further study of molecular mechanism. In addition, the comprehensive curative effect of this new method and its impact on ovarian function and fertility need to be confirmed in large multicenter randomized clinical trials.S1 TableE1/E2: EuE abtained before and after LUAO; A1/A2: EE abtained before and after LUAO.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Despite the fact that insertions/deletions (INDELs) are the second most common type of genetic variations and variable number tandem repeats (VNTRs) represent a large portion of the human genome, they have received far less attention than single nucleotide polymorphisms (SNPs) and larger forms of structural variation like copy number variations (CNVs), especially in genome-wide association studies (GWAS) of complex diseases like polygenic obesity. This is exemplified by the vast amount of review papers on the role of SNPs and CNVs in obesity, its related traits , and its related complications . Hence, this paper reviews the types of INDELs and VNTRs that have been studied for association with obesity and its related traits and complications.FTO, genes in the leptin–proopiomelanocortin pathway, and UCP2/3. Given the important role of the brain serotonergic and dopaminergic reward system in obesity susceptibility, the association of INDELs and VNTRs in these neurotransmitters’ metabolism and transport genes with obesity is also reviewed. Next, the role of INS VNTR in obesity and its related traits is questionable, since recent large-scale studies failed to replicate the earlier positive associations. As obesity results in chronic low-grade inflammation of the adipose tissue, the proinflammatory cytokine gene IL1RA and anti-inflammatory cytokine gene IL4 have VNTRs that are implicated in obesity. A systemic proinflammatory state in combination with activation of the renin–angiotensin system and decreased nitric oxide bioavailability as found in obesity leads to endothelial dysfunction. This explains why VNTR and INDEL in eNOS and ACE, respectively, could be predisposing factors of obesity. Finally, two novel genes, DOCK5 and PER3, which are involved in the regulation of the Akt/MAPK pathway and circadian rhythm, respectively, have VNTRs and INDEL that might be associated with obesity.These INDELs and VNTRs could be found in the obesity loci or genes from the earliest GWAS and candidate gene association studies, like In conclusion, INDELs and VNTRs could have important functional consequences in the pathophysiology of obesity, and research on them should be continued to facilitate obesity prediction, prevention, and treatment. The worldwide prevalence of overweight and obesity, which rose by 27.5% in adults and 47.1% in children between 1980 and 2013 [LEP) [LEPR) [POMC) [MC4R) [The study of genetic factors involved in body weight regulation in humans heavily extrapolated from studies on rodent monogenic obesity models in the 1990s. Monogenic obesity is a rare form of severe obesity that results from gene mutations that have large effect sizes. By screening children with severe, young-onset obesity for the genetic defects identified in mice, loss-of-function (LOF) mutations causing deficiencies are found in genes encoding for appetite-regulating hormones or their receptors such as leptin (LEP) , leptin ) [LEPR) , proopio) [POMC) , and mel) [MC4R) .Fortunately, after the completion of the Human Genome Project in 2003  and the Genetic variations in the human genome present as (i) single nucleotide polymorphisms (SNPs), (ii) insertions/deletions (INDELs) ranging from 1 to 10,000 bp in length , and , and genes in the leptin–proopiomelanocortin pathway and uncoupling protein families. It is possible that the association between INDELs/VNTRs and obesity is affected by the obesity-related SNPs because of strong linkage disequilibrium, and the risk or protective variants of INDELs/VNTRs are linked to the counterparts of the focal SNPs.Numerous INDELs and VNTRs are located around obesity-susceptible genes or loci identified by the earliest GWAS and candidate gene studies. These mainly include genes that are involved in energy balance, appetite regulation, and adipogenesis, i.e., fat mass and obesity-associated protein gene , makingentified . FTO is ons 2, 3 , and 8 [ons 2, 3 . By usinl INDELs . Three il INDELs .ob has been attributed to mutations in the mouse leptin gene [LEP) cause early-onset monogenic obesity in humans [LEP and its receptor LEPR, POMC and MC4R [The discovery that the mouse obesity phenotype tin gene , and than humans , 39, hasn humans , 41. A Gand MC4R .LEP cause monogenic obesity, there have been numerous research in determining whether genetic variations in or near LEP influences susceptibility to polygenic obesity. Using STR markers flanking the LEP locus at human chromosome 7q31.3-32.1, several groups reported evidence of linkage and/or association between these STRs and obesity-related traits, albeit with inconsistencies [LEP locus was extremely strong [n, was first identified at 3912 bp 3′ of the LEP stop codon (476 bp 3′ of the 3′ UTR) [LEP -2548GG SNP, LEP VNTR/SNP class I/G combined genotypes  were significantly associated with obesity and increased BMI, WC, leptin, and triglycerides in women [LEPR Gln223Arg and PRO1019pro SNPs, had elevated insulin [While mutations in stencies –47. A mey strong . A tetra 3′ UTR) . Fifteen 3′ UTR) . Neverth 3′ UTR) . In anot 3′ UTR) . In the  3′ UTR) , 53. The 3′ UTR) . In comb insulin )—allele  insulin .LEPR, located near the STR D1S200, has been associated with increased BMI and fat mass [LEPR D1S200 17 allele was associated with increased susceptibility to obesity and increased BMI, WC, and WHR in Brazilian individuals [LEPR by approximately 9 and 3 cM, such as D1S3728 and D1S1665, were proposed to contribute to plasma leptin concentrations, adiposity, and body weight in individuals with dyslipidemia [n, and 16, (CTTT)n, of LEPR were shown to be associated, respectively, with BMI and fat-free mass in the Quebec Family Study [LEPR was associated with increased body weight in patients in the Finnish Diabetes Prevention Study [LEPR (containing a putative phosphorylation site) was not associated with obesity in Dutch Caucasians [fat mass . The LEPividuals . Other Sipidemia . Two VNTly Study . A commoon Study , while aucasians .POMC had strong linkage with serum leptin levels and fat mass in Mexican Americans [POMC—D2S2170, D2S144, D2S1268, and D2S1348—showed no association with obesity in Samoans [POMC (codon 94) [A genome-wide scan found that D2S1788 GATA-tetranucleotide VNTR near mericans , 63. Thimericans , Africanmericans , and Hismericans , but notmericans . This VNmericans . However Samoans . Out of odon 94)  was assoodon 94)  men. Howodon 94) , obesityodon 94) .FTO, MC4R has been extensively studied in obesity research. To date, more than 160 mutations in MC4R, mostly heterozygous (codominant inheritance), were identified mainly in obese individuals, encompassing nonsynonymous, nonsense, deletion, and frameshift mutations. Therefore, MC4R is the most commonly known monogenic cause of human obesity . In obesity , 76. Yeo obesity . Another obesity . As the fter FTO , 80.MC4R gene—D18S851, D18S487, D18S69, D18S858, D18S849, D18S1155, D18S64, D18S38—have demonstrated linkage with obesity in Finnish sib pairs, with D18S849 showing the strongest linkage [Eight STRs flanking the  linkage . The D18 linkage . This lo linkage  and canc linkage .UCP2/UCP3 cluster have been considered candidate markers for obesity, diabetes, and fat metabolism in humans [UCP2 and UCP3 showed some evidence of linkage with obesity and BMI [UCP2–UCP3 are unlikely to have a substantial effect on the expression of obesity-related phenotypes in Mexican American [Uncoupling proteins (UCPs) are mitochondrial transporters that mediate energy homeostasis and thermogenesis. Genetic variants in the n humans . D11S912 and BMI , 81. The and BMI , consist and BMI . Campbel and BMI . HoweverAmerican  and CaucAmerican  populatiUCP2 exon 8 (reviewed in [A 45-bp INDEL variant in the 3′ UTR of iewed in ) has beeiewed in , 90. Preiewed in , 91–94. iewed in . Howeveriewed in . Similariewed in , while Wiewed in . Also, niewed in .Given the important role of the brain serotonergic and dopaminergic reward system in weight gain and obesity susceptibility, investigation of the candidate genes of these neurotransmitters’ metabolism and transport is a growing area of research , 99.SLC6A4, recycles serotonin (5-HT) after an action potential and regulates the availability of serotonin at the synaptic cleft [SLC6A4 has been a candidate gene for a variety of neuropsychiatric conditions, given the importance of serotonergic function in mood and the widespread clinical use of selective serotonin reuptake inhibitors (SSRIs) as anxiolytics and antidepressants [SLC6A4 have been studied extensively in search of association with a range of neuropsychiatric phenotypes (reviewed in [The serotonin transporter protein (SERT or 5-HTT), which is encoded by ic cleft . Since iressants . In addiiewed in ). The seiewed in . The seciewed in , 104.Z-score, sum of skinfolds, and WC) and higher food intake at the assessments of the three stages in early childhood [A study with Argentinean children/adolescents demonstrated that individuals with the 5-HTTLPR S allele were found to be at greater risk of being overweight/obese as result of a genetic predisposition that leads to higher food consumption . Anotherhildhood . The S ahildhood , but no hildhood .On the other hand, Bah et al. showed different results . They shThe first study which investigated the association between STin2 VNTR and obesity found no significant effect on obesity in Turkish adults . HoweverSLC6A3/DAT1, is a membrane-spanning protein which terminates the synaptic transmission of dopamine by reuptake into the presynaptic nerve terminals [DAT1 contains a VNTR in its 3′ UTR, consisting of repetitions of a 40-bp core sequence [The dopamine transporter (DAT1), encoded by erminals . The humsequence . This VNsequence . The 9-rsequence . The liksequence . This VNsequence . Howeversequence .More literature reported on the association between this VNTR and eating behavior. It was found that there was an association between this VNTR and binge eating behavior , suggestDRD2-141C INDEL and future weight gain, there was no association found between this INDEL and change in BMI over a 2-year follow-up [DRD2/ANKK1-TaqIA SNP—with both obesity [Obese subjects, relative to lean ones, have reduced D2 receptors (DRD2) in the striatum , due to ollow-up . Other s obesity  and addi obesity , 129 insDRD4, which is highly expressed in the prefrontal cortex and other brain regions that are involved in the reward circuits that mediate the reinforcing properties of food, such as the amygdala, hippocampus, and hypothalamus [DRD4 VNTR and higher BMI correlation was shown [Another gene encoding dopamine receptors is thalamus , 131. Ththalamus . Nine althalamus . This VNthalamus . This VNthalamus , and witthalamus . In someas shown , 138. Thas shown .DRD4 VNTR also influences BMI and body composition in the context of other environmental factors. For example, 7R allele/season-of-birth interactions increase the risk for obesity in women with either seasonal affective disorder [disorder  or bulimdisorder . A studydisorder . Childredisorder . Howeverdisorder . Similardisorder . Similardisorder . There wdisorder .MAOA located on the short arm of the X chromosome between bands Xp11.23 and Xp11.4 [MAOA VNTR alleles  with weight and BMI in Caucasian populations [Monoamine oxidase (MAO) is an outer membrane mitochondrial enzyme that catalyzes the turnover of several catecholamine neurotransmitters, namely serotonin, noradrenaline, and dopamine . MAOA, ad Xp11.4 . It contd Xp11.4 , with thd Xp11.4 . Certaind Xp11.4 , 149, whd Xp11.4 . A previulations , 151–153ulations , while Dulations  and our ulations . The inculations .TH, is located at chromosome 11p15 [n is a tetranucleotide VNTR located in the first intron of TH, giving five alleles—T6, T7, T8, T9, and T10 [n polymorphism involving the 3′ end of exon 1 and differential exclusion at exon 2 will produce an uneven regulatory effect due to the different combination of (TCAT)n repeats [TH mRNA expression was significantly decreased in the substantia nigra, ventromedial hypothalamic nucleus, and ventral tegmental area of the high-fat-diet-induced obese and obese-resistant mice compared to controls [TH expression in their peripheral blood mononuclear cells (PBMCs) compared with controls, and TH expression was also significantly negatively correlated with WC [TH VNTR was not associated with obesity, but nevertheless, subjects with the T9 allele had significantly highest SBP and visceral fat level (VFL) and lowest pulse rate.Tyrosine hydroxylase (TH) is a rate-limiting enzyme in adrenaline, noradrenaline, and dopamine synthesis, and its coding gene, me 11p15 . An exte and T10 . The alt repeats . It has  repeats , 160. Si repeats , and per repeats . Howevercontrols . Individ with WC . In our Free fatty acid accumulation in the liver, adipose tissue, and skeletal muscle of obese patients interferes with normal insulin signaling, which will lead to insulin resistance . As a coINS). INS is located between TH and the insulin-like growth factor II gene (IGF2) on 11p15.5 [INS transcription starting site (promoter region), has been largely studied in cohorts of children and adolescents [INS VNTR have been observed in Caucasians: the common short class I (26–63 repeats) and long class III (141–209 repeats) and the rare class II (about 80 repeats) [INS mRNA have been detected in the pancreas of a cadaver adult and fetus carrying class I and class III alleles, albeit with lower levels in the latter allele [A gene that may be involved in the development of metabolic syndrome is the gene codifying for insulin itself  . Differer allele , 171.INS VNTR has been intensively studied for association with glucose metabolism in a number of metabolic disorders. Initially, class I allele was reported to be associated with higher risk of type 1 diabetes in UK families [INS VNTR association with T2D were also mainly not successfully replicated [The families , and thifamilies . On the families , higher families  and infafamilies  levels, families . Howeverfamilies , 178 or families , 179. Fiplicated , 181, alplicated .INS VNTR in the genetic risk for obesity was first investigated in children. Strong evidence for a family-based association with 1.8-fold increased risk of early-onset obesity for class I allele was found, when this allele is paternally inherited [INS VNTR with body composition and insulin levels in cohorts of children were inconclusive [INS VNTR [The putative contribution of the nherited . An earlnherited . Howevernclusive –187; fornclusive  and UK pnclusive  studies nclusive , due to INS VNTR . HoweverINS VNTR .IL1RA, and to date, six distinct alleles corresponding to 1, 2, 3, 4, 5, and 6 copies of the VNTR have been identified [IL1RA VNTR and BMI in Koreans [IL1RA II allele VNTR was associated with higher TBF value and higher risk for overall adiposity [Interleukin-1 receptor antagonist (IL-1ra), also known as IL-1RN, is an endogenous competitive inhibitor of proinflammatory IL-1α and IL-1β . IL-1ra entified . The 4-rentified . This VNentified ). IL1RA entified . With re Koreans  and Nort Koreans . Similardiposity .IL1RA, IL4 has a 70-bp VNTR polymorphism within intron 3, and two common alleles are B1 and B2 that have two and three tandem repeats, respectively [IL-4, secreted by activated Th2 lymphocytes, basophils, and mast cells, executes many biological roles such as induction of Th2 differentiation, B cell proliferation, and immunoglobulin class switching . In animectively . This VNectively . Indeed,ectively . There hectively , rheumatectively , and sysectively . With reectively , 216. Ouectively . A previectively .l-arginine by nitric oxide synthase (NOS). There are at least three isoenzymes of NOS: constitutive neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and constitutive endothelial NOS (eNOS or NOS-3), located on different chromosomes and expressed in different cell lines [Nitric oxide (NO) is synthesized from ll lines . eNOS hall lines . The adill lines –222.eNOS has been reported to possess approximately 303 variations including a VNTR at intron 4, intron 13 with a CA repeat, and enormous SNPs. There are three variations which are putatively functional and studied frequently, namely Glu298Asp, 27-bp VNTR intron 4a/b, and T-786C [eNOS expression [cis-acting role in eNOS promoter function [eNOS-specific transcriptional suppression by altering histone acetylation and DNA methylation [eNOS mRNA levels [eNOS expression and reduced NO levels, which may lead to low fat oxidation over time and a mild progression of increase in body fat [d T-786C . At the d T-786C . Plasma pression , 225. Infunction . Others hylation . SpecifiA levels . Overallbody fat .eNOS SNPs are associated with T2D [eNOS SNPs also appear to increase susceptibility for hypertriglyceridemia and low HDL [eNOS VNTR allele, genotype, nor their combination with angiotensin-converting enzyme (ACE) INDEL, apolipoprotein E , and LEP G2548A presented as a risk for hypertension, elevated triglycerides, and total, LDL, or HDL cholesterol in the Roma minority population of Croatia [A pioneer study investigating the effect of this VNTR on obesity in a sample of the Tunisian population found that carriers of the (b) allele presented 1.7 times higher risk of obesity . Correlawith T2D  and insuwith T2D –234. eNO low HDL  and wors low HDL . However Croatia .ACE, defined as either the presence  or absence  of a 287-bp insert in intron 16 of the gene on chromosome 17q23 (dbSNP rs1799752) [ACE, a key enzyme in the renin–angiotensin system (RAS), converts angiotensin I into vasoconstrictor molecule angiotensin II . Body fa1799752) . This IN1799752) ), rangin1799752) , myocard1799752) , and hyp1799752)  to obesi1799752) , T2D [241799752) , and red1799752) .Research on the association of this INDEL with obesity specifically has been growing in the past two decades, mainly showing the role of the D allele or DD genotype in increased risk for overweight/obesity. The DD genotype was associated with larger increases in body weight and BP in older Italian men, as well as with higher incidence of overweight . SpanishDOCK5 which includes two VNTRs of complex sequence composition (one in 5′ UTR and another in intron 1 of DOCK5) which flank a common 3975-bp INDEL (in intron 1 of DOCK5) found a significant association of these VNTRs and INDEL with childhood and adult severe obesity [DOCK5 VNTRs and deletion was also evidenced by the association between DOCK5 transcript levels and variants in adipose tissue from a Swedish family sample [DOCK5 could contribute to obesity is currently unknown, but it has been postulated that DOCK5, a member of the DOCK family of guanine nucleotide exchange factors, could bind to protein phosphatase 2 to inactivate v-akt murine thymoma viral oncogene homolog (Akt) proteins and mitogen-activated protein kinases 1 and 3 (MAPK1 and 3), modulating the anorectic effects of leptin [In-depth investigation of a complex region on chromosome 8p21.2 encompassing  obesity . Supporty sample . The mecf leptin .PER3 defined as four repeats  or five repeats  in humans [PER3 5/5 genotype was associated with increased morning preference, earlier wake and bed time, and reduced daytime sleepiness [A 54-bp VNTR in exon 18 of n humans  has beenn humans . Particueepiness . In a sieepiness . No assoeepiness .A review paper on the role of INDELs and VNTRs in obesity is virtually nonexistent. Hence, this paper reviews the types of INDELs and VNTRs that have been studied for association with obesity and its related traits and complications, as summarized in Table Before the establishment of SNP-based GWAS genotyping platforms like SNP microarray, INDELs and VNTRs were commonly used as a genetic maker for linkage and candidate gene association analyses of polygenic traits including obesity, as evidenced by the numerous obesity sequence tagged sites (STSs) as stated in the earlier part of the review. This is because genotyping of INDELs and VNTRs is easy with low-resolution gel electrophoresis methods. Virtually, obesity loci identified in the past studies using INDELs or VNTRs were not replicated in the SNP-based GWAS with enough sample sizes. It suggests that the many of old-fashioned obesity loci were unfortunately false positive. Besides insufficient sample size, these false positive loci could also be attributed to the other reasons as outlined by Li and Meyre , which iEven if an original study describes a true positive association for a particular INDEL/VNTR, replication could be challenging, as evidenced by the discrepancies of findings in the association studies described in this review. Again, the reasons as outlined by Li and Meyre  include Some of these pitfalls in obesity loci discovery and replication genetic association studies could be overcome by following the recommendations by Li and Meyre  and the INDELs and VNTRs have significant functional consequences by regulating gene transcription, translation efficiency, and stability of mRNA or by modifying the activity of proteins by altering their structure. The final publication of the phase 3 1000 Genomes Project in October 2015, which has 3.4 million bi-allelic INDELs and 60,000 structural variants , has pro


Clostridium difficile isolates from patients in the lower Hudson Valley, New York, USA, were achieved. These isolates represent four common sequence types  belonging to two distinct phylogenetic clades. All isolates have a 4.0- to 4.2-Mb circular chromosome, and one carries a phage.Complete genome sequences of four toxigenic  Clostridium (Clostridiodes) difficile is a Gram-positive, spore-forming, anaerobic bacterium that causes antibiotic-associated diarrhea and life-threatening pseudomembranous colitis. The incidence of C. difficile infections (CDIs) has dramatically increased in the last decade. It is estimated that approximately 453,000 incident infections with approximately 29,000 deaths occurred in the United States in 2011 (C. difficile is closely linked to two major exotoxins (toxin A and toxin B) encoded by genes tcdA and tcdB, respectively, in a pathogenicity locus (PaLoc) (C. difficile can be enhanced by producing binary toxin (CDT)  ((CdtLoc) .C. difficile , only 3  were from humans in the United States. In this study, we sequenced four C. difficile clinical isolates  from patients of the lower Hudson Valley, New York, USA. These isolates were recovered from patients with symptomatic CDIs and represented the most common strains in this region. Total genomic DNA of each isolate was extracted with a Qiagen QIAamp genomic DNA kit. Whole-genome sequencing was performed on both the Pacific Bioscience (PacBio) RS II and Illumina MiSeq platforms. A sequencing library for the MiSeq platform was prepared using the Illumina Nextera XT DNA sample prep kit, and paired-end (2 × 150-bp) sequencing was performed. A library for the PacBio single-molecule real-time (SMRT) sequencing system was processed with the PacBio SMRTbell template preparation kit. Genome sequences were de novo assembled by PacBio SMRT analysis and/or the SPAdes assembler  database (http://pubmlst.org/cdifficile): R0104a (ST1), W0022a (ST2), W0003a (ST8), and W0023a (ST42). The four isolates represent the most common strains recovered in this region. MLST-based phylogeny further designated these isolates into two distinct phylogenetic clades: R0104a as clade 2, and the others as clade 1.The genomes of W0003a, W0023a, and R0104a each contain a single circular chromosome of 4,075,361, 4,110,080, and 4,190,038 bp, respectively. W0022a consists of a circular chromosome of 4,188,456 bp and a circular phage of 31,888 bp. The sequence type (ST) of each C. difficile 630 (GenBank accession number NC_009089), the genomes of all four toxigenic C. difficile isolates contain intact tcdA and tcdB genes in the PaLoc, of 7,133 and 7,101 bp, respectively. In addition, a complete CdtLoc, including intact binary toxin genes cdtA and cdtB and their regulatory cdtR, was identified in R0104a, whereas an incomplete CdtLoc, containing only the cdtR gene, was observed in isolates W0003a, W0022a, and W0023a (Compared to the reference genome d W0023a , 10.CP025044 to CP025047.Complete genome sequences of the four isolates reported here have been deposited in GenBank under the accession numbers