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Sproule, Thomas J., Robert Y. Wilpan, John J. Wilson, Benjamin E. Low, Yudai Kabata, Tatsuo Ushiki, Riichiro Abe, Michael V. Wiles, Derry C. Roopenian i John P. Sundberg. "Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum". PLOS ONE 18, nr 10 (26.10.2023): e0293218. http://dx.doi.org/10.1371/journal.pone.0293218.
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The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.
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Corà, Davide, Federico Bussolino i Gabriella Doronzo. "TFEB Signalling-Related MicroRNAs and Autophagy". Biomolecules 11, nr 7 (4.07.2021): 985. http://dx.doi.org/10.3390/biom11070985.
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The oncogenic Transcription Factor EB (TFEB), a member of MITF-TFE family, is known to be the most important regulator of the transcription of genes responsible for the control of lysosomal biogenesis and functions, autophagy, and vesicles flux. TFEB activation occurs in response to stress factors such as nutrient and growth factor deficiency, hypoxia, lysosomal stress, and mitochondrial damage. To reach the final functional status, TFEB is regulated in multimodal ways, including transcriptional rate, post-transcriptional regulation, and post-translational modifications. Post-transcriptional regulation is in part mediated by miRNAs. miRNAs have been linked to many cellular processes involved both in physiology and pathology, such as cell migration, proliferation, differentiation, and apoptosis. miRNAs also play a significant role in autophagy, which exerts a crucial role in cell behaviour during stress or survival responses. In particular, several miRNAs directly recognise TFEB transcript or indirectly regulate its function by targeting accessory molecules or enzymes involved in its post-translational modifications. Moreover, the transcriptional programs triggered by TFEB may be influenced by the miRNA-mediated regulation of TFEB targets. Finally, recent important studies indicate that the transcription of many miRNAs is regulated by TFEB itself. In this review, we describe the interplay between miRNAs with TFEB and focus on how these types of crosstalk affect TFEB activation and cellular functions.
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Yu, Miaoying, Yinghui Wei, Kui Xu, Shasha Liu, Lei Ma, Yangli Pei, Yanqing Hu i in. "EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells". PeerJ 7 (29.01.2019): e6314. http://dx.doi.org/10.7717/peerj.6314.
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Background Self-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood. Methods Here, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478. Results EGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs. Conclusions Taken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.
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Arkins, S., N. Rebeiz, A. Biragyn, D. L. Reese i K. W. Kelley. "Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts." Endocrinology 133, nr 5 (listopad 1993): 2334–43. http://dx.doi.org/10.1210/endo.133.5.8404686.
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Woodall, SM, NS Bassett, PD Gluckman i BH Breier. "Consequences of maternal undernutrition for fetal and postnatal hepatic insulin-like growth factor-I, growth hormone receptor and growth hormone binding protein gene regulation in the rat". Journal of Molecular Endocrinology 20, nr 3 (1.06.1998): 313–26. http://dx.doi.org/10.1677/jme.0.0200313.
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The mechanisms that contribute to postnatal growth failure following intrauterine growth retardation (IUGR) are poorly understood. We demonstrated previously that nutritional deprivation in the pregnant rat leads to IUGR in offspring, postnatal growth failure and to changes in endocrine parameters of the somatotrophic axis. The present study examines the effects of maternal undernutrition (30% of the ad libitum available diet; IUGR group) throughout pregnancy on hepatic insulin-like growth factor-I (IGF-I), growth hormone receptor (GHR) and GH-binding protein (GHBP) gene expression using solution hybridisation/RNase protection assays (RPAs). Animals were killed at fetal (E22, term=23 days) and postnatal (birth, days 5, 9, 15, 21) ages, livers were collected and RNA extracted for RPAs. Results demonstrate the presence of all IGF-I mRNAs resulting from transcription start sites (ss) in exon 1 (ss1/2, ss3, ss2 spliced), exon 2, the two IGF-I E-domain variants (Ea and Eb) as well as GHR and GHBP mRNAs in hepatic tissue at E22 in both the ad libitum fed and IUGR offspring. In the postnatal liver, IGF-I ss1/2, ss3, ss2 spliced, Ea and Eb IGF-I variants as well as GHR and GHBP mRNA transcripts increased in abundance from birth to day 21. IGF-I exon 2 transcripts were relatively constant from E22 until postnatal day 15, then increased at postnatal day 21 in both the ad libitum fed and IUGR offspring. The expressions of all hepatic IGF-I leader exon ss and Ea domain variants were significantly reduced in IUGR offspring (P<0.05) from E22 to postnatal day 9. In contrast, relative abundance of hepatic IGF-I Eb variants, GHR and GHBP mRNAs were unaltered in IUGR offspring compared with the ad libitum fed animals. Whether these postnatal effects of undernutrition are a direct consequence of IUGR or whether they are related, in part, to differences in postnatal food intake remains to be investigated. In summary, we have demonstrated that hepatic IGF-I ss within exon 1 and exon 2 are coordinately regulated. Use of exon 1 ss increased during normal development and decreased with IUGR without changes in GHR or GHBP gene expression. Eb transcripts, thought to represent GH-dependent endocrine regulation of IGF-I, were unchanged in IUGR. These results suggest a possible postreceptor defect in GH action as a consequence of IUGR.
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Gajardo, Gonzalo, Luis Paiva, Cesar Ulloa-Leal, Ximena Valderrama, Gerardo López, Albert Carrasco, Alejandra Isabel Hidalgo, Mauricio E. Silva, Patricio I. Palma i Marcelo H. Ratto. "Administration of Beta-Nerve Growth Factor during the Preovulatory Stage Improves Endocrine and Luteal Function in Dairy Heifers". Animals 13, nr 6 (9.03.2023): 1004. http://dx.doi.org/10.3390/ani13061004.
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The neurotrophin beta-nerve growth factor (NGF), which is present in the semen of different mammals, elicits potent ovulatory and luteotrophic actions in llamas following systemic administration. Here, we determine if purified NGF given intramuscularly (IM) during the preovulatory stage affects the corpus luteum (CL), hormone production, endometrial gene expression, and pregnancy rate of dairy heifers. Holstein-Friesian heifers were estrus-synchronized using estradiol benzoate (EB) plus an intravaginal progesterone (P4) device (DIB). After eight days, the device was removed and cloprostenol was given IM; the next day (day 9), heifers received EB IM plus one of the following: (i) 1 mg of NGF (NGF D9 group), (ii) 1 mg of NGF 32 h after EB (NGF D10 group), or (iii) phosphate buffer saline (control group). To measure pregnancy rates, heifers were treated similarly, then artificially inseminated with sexed semen 48–52 h after DIB removal, then an ultrasound was conducted 30 days after insemination. The females given NGF along with EB (NGF D9) showed significantly higher luteinizing hormone (LH) concentrations, larger CL vascular areas, and higher plasma P4 concentrations than the NGF D10 and control animals. Downregulation of the P4 receptor (PGR), and upregulation of both lipoprotein lipase (LPL) and Solute Carrier Family 6 member 14 (SLC6A14) endometrial genes, were detected in NGF D9 heifers. Furthermore, these heifers had a 10% higher pregnancy rate than the control group. We conclude that the higher P4 output, in response to the early NGF administration, led to the enhanced gene expression of transcripts related to uterine receptivity that may result in enhanced pregnancy rates.
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SKOUDY, Anouchka, Meritxell ROVIRA, Pierre SAVATIER, Franz MARTIN, Trinidad LEÓN-QUINTO, Bernat SORIA i Francisco X. REAL. "Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells". Biochemical Journal 379, nr 3 (1.05.2004): 749–56. http://dx.doi.org/10.1042/bj20031784.
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Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor β, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation.
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Schindler, Maria, Sünje Fischer, René Thieme, Bernd Fischer i Anne Navarrete Santos. "cAMP-Responsive Element Binding Protein: A Vital Link in Embryonic Hormonal Adaptation". Endocrinology 154, nr 6 (8.04.2013): 2208–21. http://dx.doi.org/10.1210/en.2012-2096.
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Abstract The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.
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Levinson-Dushnik, M., i N. Benvenisty. "Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells." Molecular and Cellular Biology 17, nr 7 (lipiec 1997): 3817–22. http://dx.doi.org/10.1128/mcb.17.7.3817.
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The transcription factors of the hepatocyte nuclear factor 3 (HNF3) family, which are active in the liver, are expressed early during endoderm differentiation. To study their involvement in early murine development, we examined their role in embryonic stem (ES) cells. HNF3alpha or HNF3beta mRNA transcripts were not detected in ES cells before differentiation, and only low levels of HNF3beta mRNA were detected at a late stage of differentiation of ES cells to embryoid bodies (EB) (20 days after induction of differentiation). To examine the consequences of overexpressing HNF3alpha or -beta in ES cells, we transfected the two genes into these cells and determined the levels of expression of tissue-specific genes during EB differentiation. Specifically, we examined expression of albumin, cystic fibrosis transmembrane conductance regulator (CFTR), phosphoenolpyruvate carboxykinase (PEPCK), alpha1-antitrypsin, transthyretin, zeta-globin, and neurofilament 68kd as markers for different cell lineages. Overexpression of HNF3beta (and to a lesser extent of HNF3alpha) induced the expression of genes associated with endodermal lineage, namely, the genes for CFTR and albumin, but did not induce the expression of genes involved in late endoderm differentiation, such as the genes for PEPCK and alpha1-antitrypsin. Moreover, expression of HNF1beta was highly induced in HNF3-overexpressing cells, while expression of HNF1alpha and HNF4 was only mildly induced in these cells. Therefore, HNF3alpha and -beta seem to be involved in early endoderm differentiation of ES cells and together with other developmental factors are apparently needed for the induction of the endodermal lineage in vivo.
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Wang, Dong, Shi-Ping Li, Jin-Sheng Fu, Sheng Zhang, Lin Bai i Li Guo. "Resveratrol defends blood-brain barrier integrity in experimental autoimmune encephalomyelitis mice". Journal of Neurophysiology 116, nr 5 (1.11.2016): 2173–79. http://dx.doi.org/10.1152/jn.00510.2016.
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The mouse autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis (MS), is primarily characterized as dysfunction of the blood-brain barrier (BBB). Resveratrol exhibits anti-inflammatory, antioxidative, and neuroprotective activities. We investigated the beneficial effects of resveratrol in protecting the integrity of the BBB in EAE mice and observed improved clinical outcome in the EAE mice after resveratrol treatment. Evans blue (EB) extravasation was used to detect the disruption of BBB. Western blot were used to detected the tight junction proteins and adhesion molecules zonula occludens-1 (ZO-1), occludin, ICAM-1, and VCAM-1. Inflammatory factors inducible nitric oxide synthase (iNOS), IL-1β, and arginase 1 were evaluated by quantitative RT-PCR (qPCR) and IL-10 by ELISA. NADPH oxidase (NOX) levels were evaluated by qPCR, and its activity was analyzed by lucigenin-derived chemiluminescence. Resveratrol at doses of 25 and 50 mg/kg produced a dose-dependent decrease in EAE paralysis and EB leakage, ameliorated EAE-induced loss of tight junction proteins ZO-1, occludin, and claudin-5, as well as repressed the EAE-induced increase in adhesion proteins ICAM-1 and VCAM-1. In addition, resveratrol suppressed the EAE-induced overexpression of proinflammatory transcripts iNOS and IL-1β and upregulated the expression of anti-inflammatory transcripts arginase 1 and IL-10 cytokine in the brain. Furthermore, resveratrol downregulated the overexpressed NOX2 and NOX4 in the brain and suppressed NADPH activity. Resveratrol ameliorates the clinical severity of MS through maintaining the BBB integrity in EAE mice.
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Song, Xu-Ting, Jia-Nan Zhang, Duo-Wei Zhao, Yu-Fei Zhai, Qi Lu, Mei-Yu Qi, Ming-Hai Lu i in. "Molecular cloning, expression, and functional features of IGF1 splice variants in sheep". Endocrine Connections 10, nr 9 (1.09.2021): 980–94. http://dx.doi.org/10.1530/ec-21-0181.
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Insulin-like growth factor 1 (IGF1), also known as somatomedin C, is essential for the regulation of animal growth and development. In many species, the IGF1 gene can be alternatively spliced into multiple transcripts, encoding different pre-pro-IGF1 proteins. However, the exact alternative splicing patterns of IGF1 and the sequence information of different splice variants in sheep are still unclear. In this study, four splice variants (class 1-Ea, class 1-Eb, class 2-Ea, and class 2-Eb) were obtained, but no IGF1 Ec, similar to that found in other species, was discovered. Bioinformatics analysis showed that the four splice variants shared the same mature peptide (70 amino acids) and possessed distinct signal peptides and E peptides. Tissue expression analysis indicated that the four splice variants were broadly expressed in all tested tissues and were most abundantly expressed in the liver. In most tissues and stages, the expression of class 1-Ea was highest, and the expression of other splice variants was low. Overall, levels of the four IGF1 splice variants at the fetal and lamb stages were higher than those at the adult stage. Overexpression of the four splice variants significantly increased fibroblast proliferation and inhibited apoptosis (P < 0.05). In contrast, silencing IGF1 Ea or IGF1 Eb with siRNA significantly inhibited proliferation and promoted apoptosis (P < 0.05). Among the four splice variants, class 1-Ea had a more evident effect on cell proliferation and apoptosis. In summary, the four ovine IGF1 splice variants have different structures and expression patterns and might have different biological functions.
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Khatua, Atanu K., Amber M. Cheatham, Etty D. Kruzel, Pravin C. Singhal, Karl Skorecki i Waldemar Popik. "Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1". American Journal of Physiology-Cell Physiology 309, nr 1 (1.07.2015): C22—C37. http://dx.doi.org/10.1152/ajpcell.00384.2014.
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The apolipoprotein L1 (APOL1) gene ( APOL1) product is toxic to kidney cells, and its G1 and G2 alleles are strongly associated with increased risk for kidney disease progression in African Americans. Variable penetrance of the G1 and G2 risk alleles highlights the significance of additional factors that trigger or modify the progression of disease. In this regard, the effect of alternative splicing in the absence or presence of G1 or G2 alleles is unknown. In this study we investigated whether alternative splicing of non-G1, non-G2 APOL1 ( APOL1 G0) affects its biological activity. Among seven APOL1 exons, exons 2 and 4 are differentially expressed in major transcripts. We found that, in contrast to APOL1 splice variants B3 or C, variants A and B1 demonstrate strong toxicity in human embryonic kidney (HEK293T) cells. Subsequently, we established that exon 4 is a major determinant of toxicity of variants A and B1 and that extracellular release of these variants is dispensable for their cytotoxicity. Although only variants A and B1 induced nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, exon 4-positive and -negative APOL1 variants stimulated perinuclear accumulation of unprocessed autophagosomes. Knockdown of endogenous TFEB did not attenuate APOL1 cytotoxicity, indicating that nuclear translocation of TFEB is dispensable for APOL1 toxicity. Our findings that a human podocyte cell line expresses exon 4-positive and -negative APOL1 transcripts suggest that these variants may play a differential role in podocyte pathology. In summary, we have identified exon 4 as a major determinant of APOL1 G0 cytotoxicity.
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Kasprzak, Aldona, i Witold Szaflarski. "Role of Alternatively Spliced Messenger RNA (mRNA) Isoforms of the Insulin-Like Growth Factor 1 (IGF1) in Selected Human Tumors". International Journal of Molecular Sciences 21, nr 19 (23.09.2020): 6995. http://dx.doi.org/10.3390/ijms21196995.
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Insulin-like growth factor 1 (IGF1) is a key regulator of tissue growth and development that is also implicated in the initiation and progression of various cancers. The human IGF1 gene contains six exons and five long introns, the transcription of which is controlled by two promoters (P1 and P2). Alternate promoter usage, as well as alternative splicing (AS) of IGF1, results in the expression of six various variants (isoforms) of mRNA, i.e., IA, IB, IC, IIA, IIB, and IIC. A mature 70-kDa IGF1 protein is coded only by exons 3 and 4, while exons 5 and 6 are alternatively spliced code for the three C-terminal E peptides: Ea (exon 6), Eb (exon 5), and Ec (fragments of exons 5 and 6). The most abundant of those transcripts is IGF1Ea, followed by IGF1Eb and IGF1Ec (also known as mechano-growth factor, MGF). The presence of different IGF1 transcripts suggests tissue-specific auto- and/or paracrine action, as well as separate regulation of both of these gene promoters. In physiology, the role of different IGF1 mRNA isoforms and pro-peptides is best recognized in skeletal muscle tissue. Their functions include the development and regeneration of muscles, as well as maintenance of proper muscle mass. In turn, in nervous tissue, a neuroprotective function of short peptides, produced as a result of IGF1 expression and characterized by significant blood-brain barrier penetrance, has been described and could be a potential therapeutic target. When it comes to the regulation of carcinogenesis, the potential biological role of different var iants of IGF1 mRNAs and pro-peptides is also intensively studied. This review highlights the role of IGF1 isoform expression (mRNAs, proteins) in physiology and different types of human tumors (e.g., breast cancer, cervical cancer, colorectal cancer, osteosarcoma, prostate and thyroid cancers), as well as mechanisms of IGF1 spliced variants involvement in tumor biology.
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Zafonte, Brian T., Tara L. Huber, Gordon Keller i Todd Evans. "Specificity of Smad Signaling during Primitive Erythropoiesis." Blood 104, nr 11 (16.11.2004): 2785. http://dx.doi.org/10.1182/blood.v104.11.2785.2785.
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Abstract Bone morphogenetic proteins (BMPs) comprise a sub-family of TGF-beta-like molecules that exert a wide range of biological activities during development, and are essential for normal hematopoiesis. However, the precise stage in development that BMP signaling regulates hematopoiesis is not defined. Three proteins, Smad1, Smad5, and Smad8 transmit BMP signals to the nucleus to activate the expression of hematopoietic-specific transcription factors. These Smads are homologous in their sequences, and appear to be regulated similarly, however their specificity in regulating hematopoiesis remains undefined. Although Smad proteins are regulated post-translationally, their expression is also under transcriptional control during development. We examined the specificity of Smad1/5/8 activity in the context of primitive erythropoiesis, using the mouse embryonic stem cell /embryoid body (ES/EB) system. We exploited ES cells with GFP targeted to the brachyury locus, in order to identify specific sub-sets of progenitors. Smad1 transcript levels are initially upregulated as ES cells become fated to mesoderm and hematopoietic progenitors, but the levels are significantly decreased in cells derived from differentiating primitive erythroid colonies. In contrast, Smad5 transcript levels show the opposite profile, being more correlated with erythroid differentiation. To directly assess the role of these Smads during erythropoiesis, their activity is being manipulated in ES cells during the commitment phases of embryonic hematopoiesis. For this purpose, inducible ES cell lines were generated capable of forcing the expression of wildtype Smad1 or Smad5, or a dominant-negative isoform of Smad5, at any stage of ES/EB development. Colony assays were used to analyze quantitatively the hematopoietic potential of these cells. Forced expression of Smad1 results in a marked increase in primitive red blood cell colony formation as compared to control ES cells. Maintenance of Smad1 expression does not appear to inhibit terminal differentiation. Based on a time-study of the induction, the effect on erythoid colonies could be due to expansion of earlier progenitors. Current experiments using the in vitro blast assay are examining the direct effect of Smad1 expression on earlier (hemangioblast) development. This data, and analogous analyses of cells induced to express Smad5 or the dominant-negative Smad isoform are in progress and will be presented. These studies should facilitate our understanding of the specificity of BMP-regulated Smads during commitment and differentiation of embryonic stem cells and hematopoietic progenitors.
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Heuston, Elisabeth F., Jens Lichtenberg, Stacie M. Anderson, Vikram R. Paralkar, Cheryl Keller Capone, Ross C. Hardison, Mitchell J. Weiss i David M. Bodine. "Differences In The Genome-Wide Epigenetic Signatures Of mRNA and Long Non-Coding RNA Genes In Mouse Erythroblasts and Megakaryocytes". Blood 122, nr 21 (15.11.2013): 1198. http://dx.doi.org/10.1182/blood.v122.21.1198.1198.
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Abstract The ENCODE project has demonstrated that epigenetic signatures, including DNA methylation and transcription factor (TF) occupancy, define gene expression. However, ENCODE was constructed using static cells that were not capable of further differentiation. We hypothesize that specific epigenetic profiles are associated with erythroid and megakaryocytic differentiation. To test this hypothesis, we isolated primary erythroblasts (EBs) and megakaryocytes (Megs) from mouse bone marrow by flow cytometry and prepared: 1) DNA for genome-wide methylation analysis using MBD2 Methyl-Seq; 2) RNA for both RNA-Seq analysis and microarray analysis of novel and annotated lncRNA expression levels; and 3) chromatin for genome-wide chromatin immunoprecipitation (ChIP-Seq) analysis of occupancy by the TFs GATA1 and NFE2. We developed the web-based high-throughput sequencing tool suite SigSeeker (http://sigseeker.org) to predict regions of methylation and TF occupancy across the genome. High-confidence methylation, GATA1, and NFE2 profiles, represented by the intersection of two independent EB and Meg biological replicates, are shown in Table 1. Of the approximate 100,000 methylated regions in EBs and Megs, 45% were shared between the two cell types. Unlike methylation, GATA1 and NFE2 occupancy showed strong cell type-specific profiles, with most GATA1 occupied sites (79%) being EB-specific, and most NFE2 occupied sites (72%) being Meg-specific. While 26% of EB-specific GATA1 peaks were co-occupied by NFE2, co-occupancy by GATA1 and NFE2 in Megs was rare (0.6%). We developed a second web-based tool called SBR (http://sbrblood.msseeker.org) to correlate ChIP-Seq and Methyl-Seq profiles with RNA-Seq and lncRNA transcriptional data sets. Almost 95% of RefSeq (coding) genes with methylation in the promoter regions were not expressed. Fewer than 5% of methylated RefSeq promoters also had TF occupancy. Unlike promoters, the bodies of RefSeq genes had a high degree of overlap between methylation and TF binding. In EBs, 38% of RefSeq genes with GATA1 occupancy in the body were methylated. Of genes with this profile, 81% were transcriptionally silent. In Megs, 42% of RefSeq genes with NFE2 occupancy in the body were methylated. However, 80% of these Meg-specific genes were transcriptionally active. In contrast to RefSeq genes, lncRNA genes have a different signature. More than 85% of EB-expressed lncRNA promoters are methylated and ∼25% of these promoters are occupied by GATA1 and/or NFE2. Over 90% of EB-expressed lncRNA gene bodies occupied by GATA1 and/or NFE2 are methylated. In contrast less than 10% of Meg-expressed lncRNA promoters are methylated and ∼only 2% of these promoters are occupied by NFE2. However, 90% of Meg-expressed lncRNA gene bodies occupied by GATA1 and/or NFE2 are methylated. Ingenuity IPA analysis of the transcriptional profiles associated with different epigenetic signatures revealed differentially regulated cellular pathways. In EBs, GATA1 occupancy in the promoters of silent genes was associated with cardiovascular (p≤ 10-7) and nervous system development (p≤ 10-7). In Megs, NFE2 promoter occupancy was associated with active genes involved in nucleic acid metabolism (p≤ 10-3) and nervous system development (p≤ 10-5). Genes expressed in both EBs and Megs were involved in transcription (p≤ 10-6), cell cycle progression (p≤ 10-5), and decreased hypoplasia (p≤ 10-20). GATA1 and NFE2 co-occupied genes expressed in both EBs and Megs were associated with suppression of bone-related (p≤ 10-6) and neuron-related transcripts (p≤ 10-8). In summary, we show that TF occupancy and methylation significantly overlap in RefSeq gene bodies, but not in promoters. These profiles have revealed that GATA1 occupancy, independent of NFE2 co-occupancy, correlates with EB-specific transcriptional silencing, whereas Meg-specific transcriptional activation is associated with NFE2 occupancy. In contrast, over 90% active and TF-occupied lncRNA (both novel and previously annotated) gene bodies are methylated. These epigenetic correlations will be important for future studies assessing the regulation of mRNAs and lncRNAs. Disclosures: No relevant conflicts of interest to declare.
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Flamant, Stephane, Jean-Claude Chomel, Christophe Desterke, Olivier Feraud, Emilie Gobbo, Maria Teresa Mitjavila-Garcia, Adlen Foudi, Frank Griscelli, Ali G. Turhan i Annelise Bennaceur-Griscelli. "Micro-RNA Profiling Reveals the Key Role of miR-206 in the Hematopoietic Potential of Human Embryonic and Induced Pluripotent Stem Cells". Blood 132, Supplement 1 (29.11.2018): 1275. http://dx.doi.org/10.1182/blood-2018-99-110729.
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Abstract Although human pluripotent stem cells (hPSCs) can theoretically be differentiated into any cell type, their ability to generate hematopoietic cells shows a major variability from one cell line to another. The reasons of this variable differentiation potential, which is constant and reproducible in a given hPSC line, are not clearly established. In order to study this phenomenon, we comparatively studied 4 human embryonic stem cell lines (hESC) and 11 human induced pluripotent stem cell (hiPSC) lines using transcriptome assays. These cell lines exhibited a significant variability to generate in vitro hematopoiesis as evaluated by day-16 embryoid body (EB) formation followed by clonogenic (CFC) assays. Four out of 11 iPSC lines (PB6, PB9, PB12.1, and PB14.3) were found to lack any hematopoietic differentiation ability whereas 7 cell lines showed variable hematopoietic potential. Among hESC lines, H9 and CL0 had low H1 and SA01 exhibited high hematopoietic potential using the above assays. Among hESC and hIPSC displaying hematopoietic potential, two sub-groups were further defined based on their hematopoietic CFC efficiency: a group of poor (generation of less than 100 CFC/105 cells, PB4 / PB10 /H9 /CL01), and high hematopoietic competency (more than 120 CFC/105 cells, PB3/ PB6.1 /PB7 /PB13 /PB17 /SA01/H1). Using global miRNome analysis performed at the pluripotency stage, the expression of 754 individual miRNAs was analyzed from 15 hPSC lines in order to explore a potential predictive marker between both sub-groups of pluripotent cells according to their hematopoietic potency. Using this approach, 27 miRNAs out of 754 appeared differentially expressed allowing the identification of a miRNA signature associated with hematopoietic-competency. The hematopoietic competency was associated with down-regulation of miR-206, miR-135b, miR-105, miR-492, miR-622 and upregulation of miR-520a, miR-296, miR-122, miR-515, miR-335. Amongst these, miR-206 harbored the most significant variation (0.04-Fold change). To explore the role of miRNA-206 in this phenomenon, we have generated a miR-206-eFGP-Puro lentiviral vector which was transfected in hESC line H1 followed by puromycin selection. As a control, H1 cell line was transfected with a Arabidopsis thaliana microRNA sequence (ath-miR-159a), which has no specific targets in mammalian cells. The correct expression of the transgenes were evaluated by flow cytometry (using GFP) and q-RT-PCR for miR-206 expression. The hematopoietic potential of H1 cell line and its miR-206-overexpressing counterpart was then tested using standard in vitro assays via d16-EB generation. We found that both CFC numbers and percentage of CD34+ were significantly lower in H1-mir-206-derived day-16 EB cells than in H1-ath- derived day-16 EB cells (p < 0.05). Thus, over-expression of miR-206 in this blood-competent hESC appeared to repress its hematopoietic potential at very early stage, since a similar lower CFC efficiency was observed in day-3 EB cells derived from miR-206 overexpressing H1 cell line. We then conducted an integrative bioinformatics analysis on miR-206 predicted target genes. To this end, 773 mRNA target transcripts of the broadly conserved (across vertebrates) miR-1-3p/206 family were identified in the TargetScan database and were integrated into the global transcriptomic analysis performed by microarray on day-16 EB cells. Using supervised ranking product analysis, 62 predicted gene targets of the miR-1-3p/206 family were found to be significantly up-regulated in hematopoietic-competent EB samples including the transcription factors RUNX1 and TAL1. Hierarchical unsupervised clustering, based on this subset of 62 predicted mir-206 target genes, fully discriminated hematopoietic-deficient from hematopoietic-competent cells. In conclusion, miRNA profiling performed at pluripotency stage could be useful to predict the ability to human iPSC to give rise to blood cell progenitors. This work emphasizes for the first time the critical role of the muscle-specific miR-206 in hematopoietic differentiation. Finally, these results suggest that genetic manipulation of hESC/iPSC could be used to enhance their hematopoietic potential and to design protocols for generation of hPSC-derived hematopoietic stem cells with long-term reconstitution ability. Disclosures No relevant conflicts of interest to declare.
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Kang, H. Y., Y. K. Choi i E. B. Jeung. "336 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING SF1 INTO THE STEROID-PRODUCING CELLS". Reproduction, Fertility and Development 27, nr 1 (2015): 256. http://dx.doi.org/10.1071/rdv27n1ab336.
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Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we obtained SF1 cDNA from mRNA of murine pituitary glands and constructed the SF1-expressing vector controlled by the CMV promoter. The SF1 transgenic mouse embryonic stem cells (SF1-mES cells) were established through transfection using the nucleofector (Lonza) and selection process using G418 at 250 μg mL–1. The SF1-mES cells were aggregated in hanging drops for 2 days and were transferred to suspension culture for an additional 1 day in mouse basal differentiation medium. Three-day-old SF1-mESC-derived EB were attached onto 6 well culture plates and differentiated into granulosa-like cells. Differentiated SF1-mES cells were analysed by expression of steroidogenesis-related genes and gonadal lineage-markers to the level of mRNA via real-time PCR method. To test the phenotype for granulosa-like cells, we confirmed transcripts of specific forkhead transcription factor FOXL2 and the follicle-stimulating hormone receptor (FSHR). On the other hand, we monitored some specific genes related to differentiation into testicular tissue. We observed the progress to primitive streak-mesendodermby gene expression analyses. In addition, we observed that differentiated SF1-mES cells express steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, and CYP19A1. Using the advanced approach, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic and gonadal lineages. We also induced granulosa-like cells. We established the effective protocol to generate ovarian cells. The derivation of these cells explores new avenues for the further study and potential application of these cells in steroidogenesis.
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Chaand, Mudit, Chris Fiore, Brian T. Johnston, Diane H. Moon, John P. Carulli i Jeffrey R. Shearstone. "Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of Nuclear Factor I X (NFIX) As a Novel Fetal Hemoglobin (HbF) Repressor". Blood 134, Supplement_1 (13.11.2019): 812. http://dx.doi.org/10.1182/blood-2019-124337.
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Human beta-like globin gene expression is developmentally regulated. Erythroblasts (EBs) derived from fetal tissues, such as umbilical cord blood (CB), primarily express gamma globin mRNA (HBG) and HbF, while EBs derived from adult tissues, such as bone marrow (BM), predominantly express beta globin mRNA (HBB) and adult hemoglobin. Human genetics has validated de-repression of HBG in adult EBs as a powerful therapeutic paradigm in diseases involving defective HBB, such as sickle cell anemia. To identify novel factors involved in the switch from HBG to HBB expression, and to better understand the global regulatory networks driving the fetal and adult cell states, we performed transcriptome profiling (RNA-seq) and chromatin accessibility profiling (ATAC-seq) on sorted EB cell populations from CB or BM. This approach improves upon previous studies that used unsorted cells (Huang J, Dev Cell 2016) or that did not measure chromatin accessibility (Yan H, Am J Hematol 2018). CD34+ cells from CB and BM were differentiated using a 3-phase in vitro culture system (Giarratana M, Blood 2011). Fluorescence-activated cell sorting and the cell surface markers CD36 and GYPA were used to isolate 7 discrete populations, with each sorting gate representing increasingly mature, stage-matched EBs from CB or BM (Fig 1A, B). RNA-seq analysis revealed expected expression patterns of the beta-like globins, with total levels increasing during erythroid maturation and primarily composed of HBB or HBG transcripts in BM or CB, respectively (Fig 1C). Erythroid maturation led to progressive increases in chromatin accessibility at the HBB promoter in BM populations. In CB-derived cells, erythroid maturation led to progressive increases in chromatin accessibility at the HBG promoters through the CD36+GYPA+ stage (Pops 1-5). Chromatin accessibility shifted from the HBG promoters to the HBB promoter during the final stages of differentiation (Pops 6-7), suggesting that HBG gene activation is transient in CB EBs (Fig 1D). Hierarchical clustering and principal component analysis of ATAC-seq data revealed that cell populations cluster based on differentiation stage rather than by BM or CB lineage, suggesting most molecular changes are stage-specific, not lineage-specific (Fig 2A, B). To identify transcription factors driving cell state, and potentially beta-like globin expression preference, we searched for DNA binding motifs within regions of differential chromatin accessibility and found NFI factor motifs enriched under peaks that were larger in BM relative to CB (Fig 2C). Transcription factor footprinting analysis showed that both flanking accessibility and footprint depth at NFI motifs were also increased in BM relative to CB (Fig 2D). Increased chromatin accessibility was observed at the NFIX promoter in BM relative to CB populations, and in HUDEP-2 relative to HUDEP-1 cell lines (Fig 2E). Furthermore, accessibility at the NFIX promoter correlated with elevated NFIX mRNA in BM and HUDEP-2 relative to CB and HUDEP-1, respectively. Together these data implicated NFIX in HbF repression, a finding consistent with previous genome-wide association and DNA methylation studies that suggested a possible role for NFIX in regulating beta-like globin gene expression (Fabrice D, Nat Genet 2016; Lessard S, Genome Med 2015). To directly test the hypothesis that NFIX represses HbF, short hairpin RNAs were used to knockdown (KD) NFIX in primary erythroblasts derived from human CD34+ BM cells (Fig 3A). NFIX KD led to a time-dependent induction of HBG mRNA, HbF, and F-cells comparable to KD of the known HbF repressor BCL11A (Fig 3B-D). A similar effect on HbF was observed in HUDEP-2 cells following NFIX KD (Fig 3E). Consistent with HbF induction, NFIX KD also increased chromatin accessibility and decreased DNA methylation at the HBG promoters in primary EBs (Fig 3F, G). NFIX KD led to a delay in erythroid differentiation as measured by CD36 and GYPA expression (Fig 3H). Despite this delay, by day 14 a high proportion of fully enucleated erythroblasts was observed, suggesting NFIX KD cells are capable of terminal differentiation (Fig 3H). Collectively, these data have enabled identification and validation of NFIX as a novel repressor of HbF, a finding that enhances the understanding of beta-like globin gene regulation and has potential implications in the development of therapeutics for sickle cell disease. Disclosures Chaand: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Johnston:Syros Pharmaceuticals: Employment, Equity Ownership. Moon:Syros Pharmaceuticals: Employment, Equity Ownership. Carulli:Syros Pharmaceuticals: Employment, Equity Ownership. Shearstone:Syros Pharmaceuticals: Employment, Equity Ownership.
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Dybko, Jaroslaw, Olga Haus, Bozena Jazwiec, Tomasz Lonc, Mateusz Sawicki i Kazimierz Kuliczkowski. "Hasford Score Is Correlated with 18 Month Molecular Response for Chronic Myeloid Leukemia Patients Treated with Second Generation Tyrosine Kinase Inhibitors and It May be Useful to Differentiate Low and Intermediate Risk Patients: A Single Institution Experience". Blood 126, nr 23 (3.12.2015): 5163. http://dx.doi.org/10.1182/blood.v126.23.5163.5163.
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Abstract BACKGROUND: Chronic myeloid leukemia (CML) has been a model disease for a variety of studies concerning scoring systems, graft versus leukemia effect or tyrosine kinase inhibitors (TKI) treatment for many years. Scoring systems playing an important role in modern medicine to establish risk-adjusted optimal therapy [1] have been always essential for CML changing treatment modalities [1-3]. The three principal risk scores : Sokal [2], Hasford [1] and European Treatment and Outcome Study (EUTOS) [3] were established in different eras of CML therapy with implications for prognosis and disease outcome [4]. Hasford metric was designed based on data of patients treated with interpheron alpha [1] and it failed to differentiate patients who achieved low and intermediate risk scores according to CCyR, MMR, and 5 years EFS [5]. However in our previous study we found Hasford score to be correlated with the long-term molecular response in patients treated with imatinib [6]. This study presents the analysis of patients treated with second generation tyrosine kinase inhibitors (2G-TKI) due to their loss of MMR on imatinib. Hasford score still distinguish patients with low and intermediate risk and correlates with 18 month molecular response. PATIENTS AND RESULTS: The original group of 88 CML patients (F/M:42/46, median age 51 (21-83), 57 low risk and 31 intermediate risk assessed by Hasford risk score) in first chronic phase without any additional chromosomal abnormalities receiving standard dose imatinib was described in our previous study [6]. Of these, 42 patients lost MMR in a median time of 47 months. Within this group we identified 20 low risk (LR) and 22 intermediate risk (IR) patients. All 42 patients were switched to 2G-TKI. The observation after 3 months of 2G-TKI treatment was also previously described. After 18 months of 2G-TKI treatment median bcr-abl transcript levels in the LR group were 0.002 (0.000-0.02) but in the IR group bcr-abl levels were 0.03 (0.000-21.1) (p=0.03, Figure 1). All 20 low risk patients achieved major molecular response (MMR). In the intermediate risk group the response rate (MMR) was approximately 73% (16/22) and there is a significant difference in a probability of achieving MMR in both groups (Fig.2, p=0.0002). CONCLUSIONS: We are aware of Hasford score limited usefulness in predicting MMR in large studies. However in our study it is still a tool to distinguish low and intermediate risk patients by their molecular response on 2G-TKI after imatinib failure. We find our results relevant to the discussion on optimizing scoring systems and first line treatment of CML patients. REFERENCES: 1. Hasford J, Pfirrmann M, Hehlmann R, Allan NC, Baccarani M, Kluin-Nelemans JC, et al. A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. Writing Committee for the Collaborative CML Prognostic Factors Project Group. Journal of the National Cancer Institute. 1998;90:850-8. 2. Sokal JE, Cox EB, Baccarani M, Tura S, Gomez GA, Robertson JE, et al. Prognostic discrimination in "good-risk" chronic granulocytic leukemia. Blood. 1984;63:789-99. 3. Hasford J, Baccarani M, Hoffmann V, Guilhot J, Saussele S, Rosti G, et al. Predicting complete cytogenetic response and subsequent progression-free survival in 2060 patients with CML on imatinib treatment: the EUTOS score. Blood. 2011;118:686-92. 4. Hu B, Savani BN. Impact of risk score calculations in choosing front-line tyrosine kinase inhibitors for patients with newly diagnosed chronic myeloid leukemia in the chronic phase. European journal of haematology. 2014;93:179-86. 5. Yahng SA, Jang EJ, Choi SY, Oh YJ, Bang JH, Park JE, Jeon HL, Lee SE, Kim SH, Byun JY, Kim DW. Comparison of Sokal, Hasford and EUTOS Scores in Terms of Long-Term Treatment Outcome According to the Risks in Each Prognostic Model: A Single Center Data Analyzed in 255 Early Chronic Phase Chronic Myeloid Leukemia Patients Treated with Frontline Imatinib Mesylate. Blood 2012;120:Abstract 2794 6. Dybko J, Medras E, Haus O, Jazwiec B, Wrobel T, Kuliczkowski K. The Hasford Score Correlates with the Long-Term Molecular Response to Imatinib Treatment for Chronic Myeloid Leukemia Patients and May be Useful for Differentiating Low and Intermediate Risk Patients: A Single Institution Experience. Blood 2014;124:Abstract 3152 Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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Hatch, Nathan D., i Scot P. Ouellette. "Identification of the alternative sigma factor regulons of Chlamydia trachomatis using multiplexed CRISPR interference". mSphere, 25.09.2023. http://dx.doi.org/10.1128/msphere.00391-23.
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ABSTRACT Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia . Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. IMPORTANCE Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia . Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.
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Singh, Bhairab N., Naoyuki Tahara, Yasuhiko Kawakami, Naoko Koyano-Nakagawa, Wuming Gong, Mary Garry i Daniel Garry. "Abstract 448: Etv2-Mir130a-Jarid2 Cascade Regulates Vascular Patterning During Embryogenesis". Arteriosclerosis, Thrombosis, and Vascular Biology 37, suppl_1 (maj 2017). http://dx.doi.org/10.1161/atvb.37.suppl_1.448.
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Remodeling of the pre-existing primitive vasculature is necessary for the formation of a complex branched vascular architecture. However, the factors that modulate these processes are incompletely defined. Previously, we defined the role of microRNAs (miRNAs) in endothelial specification. In the present study, we further examined the Etv2-Cre mediated ablation of Dicer L/L and characterized the perturbed vascular patterning in the embryo proper and yolk-sac. We mechanistically defined an important role for miR-130a , an Etv2 downstream target, in the mediation of vascular patterning and angiogenesis in vitro and in vivo . Inducible overexpression of miR-130a resulted in robust induction of vascular sprouts and angiogenesis with increased uptake of acetylated-LDL. Mechanistically, miR-130a directly regulates Jarid2 expression by binding to its 3’-UTR region. CRISPR/Cas9 mediated knockout of miR-130a showed increased levels of Jarid2 in the ES/EB system. Further, the levels of Jarid2 transcripts were increased in the Etv2-null embryos at E8.5. In the in vivo settings, injection of miR-130a specific morpholinos in zebrafish embryos resulted in perturbed vascular patterning with reduced levels of endothelial transcripts in the miR-130a morphants. qPCR and in situ hybridization techniques demonstrated increased expression of jarid2a in the miR-130a morphants in vivo . These findings demonstrate a critical role for Etv2-miR-130a-Jarid2 in vascular patterning both in vitro and in vivo .
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Shillingford, Jonathan M., i James A. Shayman. "Functional TFEB activation characterizes multiple models of renal cystic disease and loss of polycystin-1". American Journal of Physiology-Renal Physiology, 16.02.2023. http://dx.doi.org/10.1152/ajprenal.00237.2022.
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Polycystic kidney disease is a disorder of renal epithelial growth and differentiation. Transcription factor EB (TFEB), a master regulator of lysosome biogenesis and function, was studied for a potential role in this disorder. The nuclear translocation and functional responses to TFEB activation were studied in three murine models of renal cystic disease, including knockouts of folliculin, folliculin interacting proteins 1 and 2, and Pkd1 as well as in mouse embryonic fibroblasts lacking Pkd1 and 3-dimensional cultures of MDCK cells. Nuclear translocation of Tfeb characterized cystic but not non-cystic renal tubular epithelia in all three murine models as both an early and sustained response to cyst formation. The epithelia expressed elevated levels of Tfeb-dependent gene products, including cathepsin B and Gpnmb. Nuclear Tfeb translocation was observed in mouse embryonic fibroblasts lacking Pkd1, but not wild type fibroblasts. Pkd1 knockout fibroblasts were characterized by increased Tfeb dependent transcripts, lysosomal biogenesis and repositioning, and increased autophagy. The growth of MDCK cell cysts was markedly increased following exposure to the TFEB agonist, compound C1, and nuclear Tfeb translocation was observed in response to both forskolin and compound C1 treatment. Nuclear TFEB also characterized the cystic epithelia but not non-cystic tubular epithelia in human ADPKD patients. The non-canonical activation of TFEB is characteristic of cystic epithelia in multiple models of renal cystic disease including those associated with the loss of Pkd1. Nuclear TFEB translocation is functionally active in these models and may be a component of a general pathway contributing to cystogenesis and growth.
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Wang, Caixia, Rui Peng, Min Zeng, Zhenhua Zhang, Shengpeng Liu, Dan Jiang, Yuanyuan Lu i Fangdong Zou. "An autoregulatory feedback loop of miR-21/VMP1 is responsible for the abnormal expression of miR-21 in colorectal cancer cells". Cell Death & Disease 11, nr 12 (grudzień 2020). http://dx.doi.org/10.1038/s41419-020-03265-4.
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AbstractMircoRNA-21 (miR-21) was found to be highly expressed in various solid tumors, and its oncogenic properties have been extensively studied in recent years. However, the reason why miR-21 is highly expressed in various tumors remains elusive. Here, we found that the expression of miR-21 was negatively correlated with the expression of vacuole membrane protein-1 (VMP1) in colorectal cancer. Transcription of VMP1 activated either by small activating RNA (saRNA) or transcriptional activator GLI3 inhibited miR-21 expression through reducing its transcripts of VMP1-miR-21 and pri-miR-21, while no significant change in miR-21 expression after exogenous overexpression VMP1 in colorectal cancer cell HCT116. Considering the overlapping location of VMP1 and miR-21 gene in genome, the result suggested that the transcription of miR-21 was inhibited by the endogenous transcriptional activation of VMP1. Furthermore, we identified that miR-21 inhibited the activation and nuclear translocation of transcription factor EB (TFEB) through reducing the inhibitory of PTEN on AKT phosphorylation, which can directly activate the transcription of VMP1. Loss of miR-21 significantly increased VMP1 expression, which could be blocked by PTEN inhibitor (VO-Ohpic) or TFEB siRNA. These results showed that miR-21 negatively regulated VMP1 transcription through the PTEN/AKT/TFEB pathway, and TFEB-induced transcriptional activation of VMP1 could inhibit miR-21 expression, thus forming a feedback regulatory loop of miR-21/VMP1. We further found that disrupting the miR-21/VMP1 feedback loop will decrease the expression of miR-21, reduce the malignancy, and increase their sensitivity to 5-fluorouracil in colorectal cancer cells. Taken together, our results revealed a novel regulatory mechanism of miR-21 expression, and targeting the miR-21/VMP1 feedback loop may provide a new approach to inhibit miR-21 expression in colorectal cancer cells.