RESUMO
The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases. Stem Cells 2017;35:725-738.
Assuntos
Processamento Alternativo/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Regeneração , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Hipertrofia , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/metabolismo , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Células Satélites de Músculo Esquelético/metabolismo , Serina/metabolismoRESUMO
Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with ß-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aß fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conjugated structure and H-donor/acceptor substituents on the aromatic rings showed to be the sine qua non structural features to provide interaction and disaggregation activity even at very low incubation time (2h). Computational simulations proved that upon binding the ligands modify the conformational dynamics and/or interact with the amyloidogenic region of the protofibril facilitating disaggregation. Significantly, in vitro results on hippocampal cells pointed out protection against glutamate toxicity and safety when administered at low concentrations (1⯵M). On the overall, in view of its higher stability in physiological conditions with respect to curcumin, of his rapid binding to fibrillar aggregates and strong depolymerizing activity, phtalimmide derivative K2F21 appeared a good candidate for both AD diagnostic and therapeutic purposes.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Curcumina/farmacologia , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Curcumina/síntese química , Curcumina/química , Relação Dose-Resposta a Droga , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.
Assuntos
Fator de Ligação a CCAAT/fisiologia , Replicação do DNA/genética , Animais , Fator de Ligação a CCAAT/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , DNA/metabolismo , Células HCT116 , Humanos , Regiões Promotoras Genéticas , Fase S/genética , Elongação da Transcrição Genética , Transcrição Gênica , Xenopus laevisRESUMO
The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.
Assuntos
Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Isoformas de Proteínas/genética , Animais , Fator de Ligação a CCAAT/biossíntese , Proliferação de Células/genética , Ciclina B/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Mioblastos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: the restrictive measures that were adopted during three waves of the COVID-19 pandemic had an impact on both the emotional state and lifestyle of the general population. We evaluated the impact of COVID-19 pandemic on lifestyles and emotional states of women planning assisted reproductive technology (ART), and whether these changes affected ART outcomes. METHODS: quantitative research, using a web-based survey, was performed on 289 Caucasian women. RESULTS: In preconception, we observed higher percentage of women with positive obstetric outcomes who reduced body weight (52.4% vs. 27.2%, p = 0.09). Over 60% of women with positive outcomes practiced physical activity vs. 47% of women with negative outcomes (p = 0.03), as well as having better quality of sleep (45% vs. 35%), and a more solid relationships with their partners (65.1% vs. 51.7%, p = 0.03). Women who increased their intake of whole grains, fruits, vegetables, and legumes (p < 0.05), according to the Mediterranean diet, showed positive outcomes. We observed that participants who experienced "very much" or "extreme" anxiety, sadness, and fear (p < 0.05) during pandemic were clearly more numerous in the group with negative pregnancy outcomes. CONCLUSIONS: healthy lifestyle together with a positive emotional state in preconception can positively influence the obstetric outcomes after ART.
RESUMO
The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokines.
Assuntos
Citocinas/fisiologia , Proteínas de Drosophila/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Proteínas de Drosophila/imunologia , Drosophila melanogaster , Reação em Cadeia da PolimeraseRESUMO
Background: The COVID-19 pandemic has been shown to impact the lifestyle of couples of reproductive age and, in particular, their desire for parenthood. The purpose of this study was to carry out an evaluation on the potential changes of desire for parenthood among infertile couples waiting for assisted reproduction during the pandemic. Methods: In this multicenter cross-sectional study, the quality of sexual life in Italian infertile couples was assessed and their well-being was evaluated before the pandemic and during the quarantine. All couples were asked to fill out a questionnaire, in which their desire for parenthood, sexual life, and well-being were investigated. Results: Out of 1650 cases, 300 patients were finally enrolled. COVID-19 negatively impacted the well-being of individuals, leading to significantly reduced scores of happiness, feeling energetic, and interest in life (p<0.05). Although most couples had prolonged infertility, a small number of cases (4.0%) achieved a spontaneous natural pregnancy during the lockdown, probably due to more intimacy and longer time spent together. However, major concerns about the consequential effects of the virus on pregnancy and the risk of contagion in the hospital led a small number of infertile couples (5.0%) to decide to postpone their parenting project. Conclusion: The COVID-19 pandemic may have created a further negative impact on couples, reducing their desire for parenthood. This attitude could result in a decrease in births in the near future.
RESUMO
The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proliferação de Células , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Isoformas de Proteínas/genética , Regeneração/genéticaRESUMO
Background: The last two decades have seen a growing number of pregnancies in women who needed the donation of oocytes. With oocyte donation pregnancies, studies on obstetric outcomes among these women revealed an increased incidence of pre-eclampsia and pregnancy-induced hypertension. Furthermore, several studies have found a higher incidence of low birth weight, preterm birth, and delivery by cesarean section in oocyte donation rather than in women subjected to assisted reproduction techniques (ART) with autologous oocytes. Numerous studies have also shown a deep connection between cardiovascular and thrombotic risk factors and adverse pregnancy outcomes. In this setting, to strictly assess the preconceptional risk for women who undergo egg donation to achieve pregnancy, the aim of our study is to draw a detailed assessment of the vascular risk profile of patients with gamete donation ART indications through the evaluation of comorbidities and cardiometabolic and thrombophilic markers Materials and Methods: Patients undergoing ART with oocyte or sperm donation or double donation of gametes underwent a careful clinical assessment through a detailed personal and family anamnesis and they were evaluated for cardiometabolic and thrombophilic profile. Clinical and demographic characteristics, comorbidities, and biohumoral parameters were collected. The study was approved by the Regional Ethical Committee(Em 2018-017 CINECA 10189). Results: We evaluated 525 women. Around 73.1% were >40 years and 35% of them were older than 45 years. There was a high prevalence of dyslipidemias (58.1%), smoking habit (24.6%), a body mass index >25 in 28.6% of patients, a high abdominal circumference in 58.1% of cases, a prevalence of acquired thrombophilia in about 7% and hereditary of 19.2%. Around 39.2% of patients had total cholesterol >200 mg/dL, 19.5% had high-density lipoprotein <48 mg/dL and 43.6% had low-density lipoprotein >115 mg/dL, and 6.9% had triglyceride values >150 mg/dL. Conclusions: A careful assessment of the preconceptional status of patients undergoing ART programs with oocyte donation can be highly recommended.
Assuntos
Doação de Oócitos , Nascimento Prematuro , Cesárea/efeitos adversos , Feminino , Fertilização in vitro , Humanos , Recém-Nascido , Doação de Oócitos/efeitos adversos , Gravidez , Resultado da Gravidez , Estudos RetrospectivosRESUMO
One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.
Assuntos
Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico/metabolismo , Desenvolvimento Muscular/imunologia , Músculo Esquelético/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular , Células HeLa , Humanos , Músculo Esquelético/citologia , Transfecção , Receptor de Lamina BRESUMO
The transcription factor NF-Y is a trimer with histone-like subunits that binds and activates CCAAT-containing promoters. NF-Y controls the expression of several key regulators of the cell cycle. In this study, we examined the functional and molecular effects of NF-YB knockdown. Cell cycle progression is affected with a G2/M-specific depletion. This is due to the inability of activation of G2/M-specific genes, as evidenced by expression profiling, RT-PCR and ChIP data. Surprisingly, apoptosis is also observed, with Caspase 3/7/8 cleavage. A role of p53 and Bcl-2 family members is important. NF-YB inactivation is sufficient to functionally activate p53, in the absence of DNA damage. Failure to maintain a physiologic level of CCAAT-dependent transcription of anti-apoptotic genes contributes to impairment of Bax/Bcl-2 and Bax/Bcl-X(L) ratios. Our data highlight the importance of fine balancing the NF-Y-p53 duo for cell survival by (i) maintaining transcription of anti-apoptotic genes and (ii) preventing p53 activation that triggers the apoptotic cascade.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Fator de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fator de Ligação a CCAAT/antagonistas & inibidores , Fator de Ligação a CCAAT/genética , Caspases/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Humanos , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
INTRODUCTION: Infertility may depend up to 27% of couples on both partners. In patients with obstructive azoospermia, testicular fine-needle aspiration represents a good option to retrieve spermatozoa, in order to perform an assisted reproductive treatment. In vitro maturation of testicular spermatozoa could be the better choice of treatment in view of the increased motility, improving fertilization and pregnancy rates. CASE DESCRIPTION: A 34-year-old azoospermic man and his 33-year-old partner referred for treatment of simultaneous male and female infertility factor. The woman presented a diminished ovarian reserve, with serum follicle stimulating hormone value of 27.15 IU/L. The man underwent trans-rectal and testicular ultrasounds that detected the congenital absence of proximal vas deferens on the right side and the absence of seminal vesicle and distal vas deferens on the left side. We proposed a chance to have their own biological child. The man underwent modified testicular fine-needle aspiration using a 18-gauge butterfly needle. Sperm retrieval was successful with 0.001 × 106 spermatozoa/mL and absence of motility. Testicular sperm suspension was cultured for 24 h to identify sperm viability, achieving 10% of sperm motility. Two metaphase II oocytes were retrieved and processed with intracytoplasmic sperm injection. Clinical pregnancy with live birth was obtained. CONCLUSION: Performing modified testicular fine-needle aspiration increases successful sperm retrieval. Testicular sperm in vitro culture for 24 h proved to be a real and practical technique to increase sperm motility, in order to select mature and viable spermatozoa and improve successful intracytoplasmic sperm injection outcomes.
Assuntos
Infertilidade , Recuperação Espermática , Espermatozoides/fisiologia , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Agulhas , Gravidez , Recuperação Espermática/instrumentação , TestículoRESUMO
BACKGROUND: The purpose of this study is to assess the impact of paternal age on the oocyte-donation outcomes in intracytoplasmic sperm injection (ICSI) cycles. METHODS: Two hundred and seventy-eight infertile couples were retrospectively involved. Inclusion criteria were: infertility from almost 1 year, normal or sub-fertile seminal parameters, overall oocyte survival rate greater than 85%. Baseline characteristics included male age, recipient age, male body mass index (BMI), smoking, drinking status. Main outcome measures: fertilization rate (FR), cleavage rate (CR), pregnancy rate (PR). RESULTS: Patients were categorized in group 1 ≤45, group 2 >45 years. A total of 1,724 frozen oocytes were included. After warming, 1,642 oocytes survived. Median overall oocyte survival rate was 100% [interquartile range (IQR), 85-100%]. Median male age was 44±5.60 years (IQR, 31-70 years). Median recipients age was 42±3.62 years (IQR, 29-50 years). Group 1 included 166 men, group 2 112 men. Two hundred and seventy-eight fresh ICSI cycle were performed. "Two-pronuclear" (2PN) FR was 72.6%±0.20%, CR 93.0%±0.16%, PR 39.6%. Miscarriage rate was 25.5%. Live birth rate per cycles was 29.5%. Comparison between group 1, group 2 and ICSI outcomes confirmed an association with FR, resulting 80.0% (IQR, 67.0-83.0%) and 67.0% (IQR, 50.0-80.0%), respectively (P<0.01). There were no significant differences between the two groups with respect to seminal parameters. CONCLUSIONS: It would be recommended more emphasis on the advancing male age when counselling older couples who undergo egg donation program.
RESUMO
In response to DNA damage, p53 activates G(1)/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G(2)/M transition. Like most G(2)/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the alphaC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)-HDAC1 before HDAC4 and HDAC5-and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G(2)/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G(2)/M transition, and (iii) delineate a new role for PCAF in cell cycle control.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Dano ao DNA/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Repressoras/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Acetilação , Acetiltransferases/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Imunoprecipitação da Cromatina , Dano ao DNA/genética , Regulação para Baixo , Fase G2 , Histona Acetiltransferases , Histona Desacetilases/metabolismo , Histona Desacetilases/fisiologia , Humanos , Camundongos , Mutação/genética , Regiões Promotoras Genéticas/genética , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fatores de Transcrição de p300-CBPRESUMO
The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.
Assuntos
Fator de Ligação a CCAAT/genética , Lamina Tipo A/genética , Neoplasias/genética , Transcrição Gênica , Regulação da Expressão Gênica , Células HCT116 , Humanos , Espectrometria de Massas , Neoplasias/patologia , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NF-YA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Transformação Celular Viral/fisiologia , Proteínas de Ligação a DNA/biossíntese , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Oncogênicas Virais/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Células HeLa , Papillomavirus Humano 18 , Humanos , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/metabolismo , Ativação TranscricionalRESUMO
OBJECTIVE: Evaluation of frontal vault symmetry and progressive facial symmetrization in a cohort of patients with hemicoronal single suture synostosis treated with a standardized cranioplasty and rigid fixation. PATIENTS AND METHODS: Fifty-four patients with hemicoronal synostosis operated between 1999 and 2014 were reviewed retrospectively. Pre, immediately postoperative and yearly photographs from the top of the skull and frontal views of the face were taken with the same head position and projection. A photogrammetric method was applied to quantify the pre and postoperative contour changes. The anterior skull hemispheres were traced, divided into two equal parts and the areas were compared. Angular measurements obtained by the intersection of the interpupillary line and the glabella perpendicular vertical line were calculated. The average photographic follow-up was 6.8 years. Range 1-14 years. RESULTS: The average advancement on the affected side was 18 mm (range: 16-23 mm). The pre-surgical cranial area on the affected side was increased on average 14.6 + 2.4% (range: 10-18%). The angular measurements documented the frontal symmetry obtained and the progressive improvement of facial symmetry. CONCLUSION: Cranioplasty with rigid fixation achieved a stable correction of anterior plagiocephaly leading to subsequent symmetrical facial growth. Photogrammetry allowed fora quantitative long-term validation.
Assuntos
Craniossinostoses/cirurgia , Face/anatomia & histologia , Osso Frontal/cirurgia , Órbita/cirurgia , Fotogrametria , Suturas Cranianas/cirurgia , Estética , Face/diagnóstico por imagem , Feminino , Seguimentos , Humanos , Lactente , Masculino , Procedimentos de Cirurgia Plástica/métodos , Estudos RetrospectivosRESUMO
The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.
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In clinical practice lung ultrasound (LUS) is becoming an easy and reliable noninvasive tool for the evaluation of dyspnea. The aim of this study was to assess the accuracy of nurse-performed LUS, in particular, in the diagnosis of acute cardiogenic pulmonary congestion. We prospectively evaluated all the consecutive patients admitted for dyspnea in our Medicine Department between April and July 2014. At admission, serum brain natriuretic peptide (BNP) levels and LUS was performed by trained nurses blinded to clinical and laboratory data. The accuracy of nurse-performed LUS alone and combined with BNP for the diagnosis of acute cardiogenic dyspnea was calculated. Two hundred twenty-six patients (41.6% men, mean age 78.7â±â12.7 years) were included in the study. Nurse-performed LUS alone had a sensitivity of 95.3% (95% CI: 92.6-98.1%), a specificity of 88.2% (95% CI: 84.0-92.4%), a positive predictive value of 87.9% (95% CI: 83.7-92.2%) and a negative predictive value of 95.5% (95% CI: 92.7-98.2%). The combination of nurse-performed LUS with BNP level (cut-off 400âpg/mL) resulted in a higher sensitivity (98.9%, 95% CI: 97.4-100%), negative predictive value (98.8%, 95% CI: 97.2-100%), and corresponding negative likelihood ratio (0.01, 95% CI: 0.0, 0.07). Nurse-performed LUS had a good accuracy in the diagnosis of acute cardiogenic dyspnea. Use of this technique in combination with BNP seems to be useful in ruling out cardiogenic dyspnea. Other studies are warranted to confirm our preliminary findings and to establish the role of this tool in other settings.
Assuntos
Dispneia/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Diagnóstico de Enfermagem , Doença Aguda , Idoso , Dispneia/diagnóstico , Feminino , Cardiopatias/complicações , Humanos , Masculino , Peptídeo Natriurético Encefálico/sangue , Valor Preditivo dos Testes , Estudos Prospectivos , Ultrassonografia/métodos , Ultrassonografia/normasRESUMO
BACKGROUND: Maxillary distraction osteogenesis (DO) in cleft lip and palate patients has been described by several authors, but most studies have a relatively short follow-up and do not clearly separate growing patients from non-growing patients. METHOD: The records of 22 consecutive patients affected by cleft lip and palate, who underwent Le Fort I osteotomy and maxillary distraction with a rigid external distractor (RED), were reviewed. The sample was subdivided into a growing and a non-growing group. All patients had pre-DO cephalometric records, immediately post DO, 12 months post DO and long-term records with a long-term follow-up of >5 years (range 5-13 years). As a control sample for the growing group, cleft children with a negative overjet not subjected to distraction or any protraction treatment during growth were followed up until the completion of growth. RESULTS: The average maxillary advancement in the growing group was 22.2 ± 5.5 mm (range: 15-32 mm); in the non-growing group, it was 17.7 ± 6.6 mm (range: 6-25 mm). Excellent post-surgical stability was recorded in the adult sample. On the other hand, growing children had an average 16% relapse in the first year post DO and an additional 26% relapse in the long-term follow-up. CONCLUSIONS: This study seems to point out that early Le Fort I DO allows for the correction of very severe deformities. It is followed by a relatively high amount of true skeletal relapse in children with cleft lip and palate. Prognosis should be discussed in depth with the family and true aesthetic and psychological needs assessed.