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1.
Genes Dev ; 34(5-6): 428-445, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32001510

RESUMO

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.


Assuntos
Núcleo Celular/patologia , Senescência Celular/fisiologia , Cromatina/patologia , Citoplasma/patologia , Mitocôndrias/patologia , Transdução de Sinais , Animais , Núcleo Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Inflamação/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
2.
PLoS One ; 14(7): e0219006, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329606

RESUMO

Transformation in chromatin organization is one of the most universal markers of carcinogenesis. Microscale chromatin alterations have been a staple of histopathological diagnosis of neoplasia, and nanoscale alterations have emerged as a promising marker for cancer prognostication and the detection of predysplastic changes. While numerous methods have been developed to detect these alterations, most methods for sample preparation remain largely validated via conventional microscopy and have not been examined with nanoscale sensitive imaging techniques. For these nanoscale sensitive techniques to become standard of care screening tools, new histological protocols must be developed that preserve nanoscale information. Partial Wave Spectroscopic (PWS) microscopy has recently emerged as a novel imaging technique sensitive to length scales ranging between 20 and 200 nanometers. As a label-free, high-throughput, and non-invasive imaging technique, PWS microscopy is an ideal tool to quantify structural information during sample preparation. Therefore, in this work we applied PWS microscopy to systematically evaluate the effects of cytological preparation on the nanoscales changes of chromatin using two live cell models: a drug-based model of Hela cells differentially treated with daunorubicin and a cell line comparison model of two cells lines with inherently distinct chromatin organizations. Notably, we show that existing cytological preparation can be modified in order to maintain clinically relevant nanoscopic differences, paving the way for the emerging field of nanopathology.


Assuntos
Carcinogênese/patologia , Cromatina/patologia , Técnicas Histológicas/métodos , Linhagem Celular , Cromatina/ultraestrutura , Etanol , Fixadores , Células HeLa , Humanos , Microscopia/métodos , Nanotecnologia , Preservação Biológica , Análise Espectral/métodos , Fixação de Tecidos/métodos
3.
Adv Exp Med Biol ; 1166: 149-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31301051

RESUMO

Cryopreservation is a technique that can keep sperm alive indefinitely, enabling the conservation of male fertility. It involves the cooling of semen samples and their storage at -196 °C in liquid nitrogen. At this temperature all metabolic processes are arrested. Sperm cryopreservation is of fundamental importance for patients undergoing medical or surgical treatments that could induce sterility, such as cancer patients about to undergo genotoxic chemotherapy or radiotherapy, as it offers these patients not only the hope of future fertility but also psychological support in dealing with the various stages of the treatment protocols.Despite its importance for assisted reproduction technology (ART) and its success in terms of babies born, this procedure can cause cell damage and impaired sperm function. Various studies have evaluated the impact of cryopreservation on chromatin structure, albeit with contradictory results. Some, but not all, authors found significant sperm DNA damage after cryopreservation. However, studies attempting to explain the mechanisms involved in the aetiology of cryopreservation-induced DNA damage are still limited. Some reported an increase in sperm with activated caspases after cryopreservation, while others found an increase in the percentage of oxidative DNA damage. There is still little and contradictory information on the mechanism of the generation of DNA fragmentation after cryopreservation. A number of defensive strategies against cryoinjuries have been proposed in the last decade. Most studies focused on supplementing cryoprotectant medium with various antioxidant molecules, all aimed at minimising oxidative damage and thus improving sperm recovery. Despite the promising results, identification of the ideal antioxidant treatment method is still hampered by the heterogeneity of the studies, which describe the use of different antioxidant regimens at different concentrations or in different combinations. For this reason, additional studies are needed to further investigate the use of antioxidants, individually and in combination, in the cryopreservation of human sperm, to determine the most beneficial conditions for optimal sperm recovery and preservation of fertility.


Assuntos
Cromatina , Criopreservação , Preservação do Sêmen , Cromatina/química , Cromatina/patologia , Criopreservação/métodos , Criopreservação/normas , Crioprotetores , Fragmentação do DNA , Humanos , Masculino , Preservação do Sêmen/métodos , Preservação do Sêmen/normas , Espermatozoides/patologia
4.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180045

RESUMO

Recent research has shown that cell-free chromatin (cfCh) particles that are released from the billions of cells that die in the body everyday can enter into healthy cells, integrate into their genomes and induce dsDNA breaks and apoptotic responses. Genomic integration of cfCh activates NF κ B suggesting a novel mechanism of induction of systemic inflammation. Since DNA damage and inflammation are underlying pathologies in multiple devastating acute and chronic disease conditions, the discovery of agents that can inactivate cfCh may provide therapeutic possibilities.


Assuntos
Anti-Inflamatórios/farmacologia , Cromatina/patologia , Histonas/genética , NF-kappa B/genética , Animais , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Transporte Biológico , Cromatina/química , Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Desoxirribonuclease I/farmacologia , Espaço Extracelular/química , Regulação da Expressão Gênica , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Inflamação/prevenção & controle , Células Jurkat/transplante , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Resveratrol/análogos & derivados , Resveratrol/farmacologia , Transdução de Sinais
5.
Fertil Steril ; 112(1): 46-53.e2, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31043234

RESUMO

OBJECTIVE: To determine whether high DNA stainability (HDS), as assessed by the sperm chromatin structure assay (SCSA), predicts the risk of early miscarriage after in vitro fertilization with intracytoplasmic sperm injection (IVF-ICSI). DESIGN: Retrospective cohort study of consecutive pregnancies after IVF and ICSI treatment. SETTING: Reproductive medicine center. PATIENT(S): A total of 1,602 pregnancies after 832 IVF and 770 ICSI treatments. INTERVENTION(S): HDS measured using SCSA. MAIN OUTCOME MEASURE(S): Early miscarriage (≤12 weeks). RESULT(S): The HDS represents the proportion of immature spermatozoa lacking the normal exchange of histone for protamine-complexed DNA, and the outcome parameter was early miscarriage (≤12 weeks). For all treatments, the odds ratio (OR) and 95% confidence interval (CI) for early miscarriage was 1.41 (1.07-1.85) if HDS >15% compared with HDS ≤15%. When comparing the two HDS categories, for ICSI, the OR was 1.44 (1.01-2.04) whereas for IVF the results were not statistically significant. CONCLUSION(S): There is a small but increased risk of early miscarriage if HDS >15% compared with HDS ≤15%. This increased risk is seen only after ICSI, not after IVF. These findings suggest that HDS can be used as a predictor of an increased risk of miscarriage in ICSI treatments.


Assuntos
Aborto Espontâneo/etiologia , Cromatina/patologia , Fragmentação do DNA , Infertilidade Feminina/terapia , Análise do Sêmen/métodos , Injeções de Esperma Intracitoplásmicas/efeitos adversos , Espermatozoides/patologia , Aborto Espontâneo/diagnóstico , Adulto , Diagnóstico Precoce , Feminino , Fertilidade , Humanos , Infertilidade Feminina/patologia , Infertilidade Feminina/fisiopatologia , Masculino , Valor Preditivo dos Testes , Gravidez , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
6.
Andrologia ; 51(8): e13316, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31099901

RESUMO

Men affected with idiopathic infertility often display basic spermiogramme values similar to fertile individuals, questioning the diagnostic impact of the World Health Organization (WHO) thresholds used. This study explored sperm DNA fragmentation in single ejaculates from 14 fertile donors and 42 patients with idiopathic infertility providing semen for assisted reproductive techniques in a university fertility clinic. Each ejaculate was simultaneously studied for sperm DNA fragmentation by the flow cytometer-based sperm chromatin structure analysis (SCSA) and the new light-microscopy-based sperm chromatin dispersion assay (SCD-HaloSpermG2® ), before and after sperm selection for in vitro fertilisation with a colloid discontinuous gradient. The WHO semen variables did not differ between groups, but DNA fragmentation after SCSA (DFI) or SCD (SDF) was significantly (p < 0.05) higher in patients (DFI: 40.2% ± 3.0 vs. SDF: 40.3% ± 1.4) than in fertile donors (DFI: 17.1% ± 2.1 vs. SDF: 20.9% ± 2.5). Sperm selection led to lower proportions of DNA-fragmented spermatozoa (DFI: 11.9 ± 1.7 vs. SCD: 10.0 ± 0.9, p < 0.05). The techniques output correlated highly and significantly (r2  = 0.82). DNA fragmentation is confirmed as a relevant variable for scrutinising patients with idiopathic infertility, beyond the evidently insufficient WHO semen analyses. Since both techniques yielded similar results, the reduced necessity of complex equipment when running SCD ought to be considered for a clinical setting.


Assuntos
Cromatina/patologia , Fragmentação do DNA , Infertilidade Masculina/patologia , Análise do Sêmen/métodos , Espermatozoides/patologia , Adulto , Estudos de Coortes , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/terapia , Masculino , Técnicas de Reprodução Assistida
7.
Breast J ; 25(3): 434-438, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30972846

RESUMO

Fine needle aspiration cytology has long been an accepted diagnostic modality in combination with physical examination and mammography to investigate breast lesions. In the present era, more proficient methods such as stereotactic mammographically guided breast biopsy is the preferred choice, however, in low resource setting, FNAC still remains the most cost effective and sampling modality to diagnose breast cancer. With the intention to evaluate the efficacy and limitations of FNAC in evaluation of breast lesions in low resource setting, we employed the Masood's cytological staging system to stratify the breast lesions and correlate them with histopathology wherever possible. All breast lesions aspirates were analyzed and classified according to the Masood's cytological scoring system and correlated with histopathological findings wherever adequate material was available. A total of 776 patients were studied of which 23 aspirates were unsatisfactory, 120 aspirates were categorized as inflammatory breast disease. Six hundred and thirty-three cases were classified according to Masood's cytological system. Nonproliferative breast diseases (Group I) encompassed maximum cases with 55% followed by carcinoma in situ and invasive cancers (Group IV) with 39% and proliferative breast disease without atypia (Group II) and with atypia (Group III) which had equal number of cases constituting 2.4% each. Cyto-histopathological correlation done in 102 cases revealed 100% concordance in group IV and 75% concordance in group III while it could not be performed in Group I and II as no histopathological specimen was available in those patients. Masood Cytological grading for breast aspirates is a reliable and an easily reproducible system which can be used to formulate appropriate treatment protocols in cases presenting with breast lesions.


Assuntos
Doenças Mamárias/patologia , Neoplasias da Mama/patologia , Citodiagnóstico/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Criança , Cromatina/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
8.
Zygote ; 27(2): 69-77, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30834849

RESUMO

SummaryDirect swim-up procedure is widely used to separate the motile competent spermatozoa from the antioxidant-rich semen. Subsequently, spermatozoa become more vulnerable to reactive oxygen species (ROS) due to their cytological characteristics. The effect of vitamin C, a highly concentrated antioxidant in the semen, on direct swim-up-enriched sperm population is not fully investigated. Therefore, the aim of the present study was to assess the effect of vitamin C on sperm functional properties during direct swim-up procedure. Semen samples were collected from 22 participants. Each semen sample was divided into several aliquots. The first portion was overlaid with sperm medium without ascorbic acid (0 µM AA). The second and third fractions were overlaid with sperm medium supplemented with 300 µM and 600 µM AA; respectively. After 1 h of incubation, basic sperm parameters, intracellular ROS levels, acrosome reaction, chromatin integrity, and glucose uptake were assessed. Swim-up without AA significantly increased the percentage of ROS(+) spermatozoa compared with the raw semen (P<0.01). Interestingly, swim-up with 300 µM AA did not increase the percentage of ROS(+) sperm compared with the raw semen. In parallel, the percentage of sperm with altered chromatin integrity was significantly lower in the 300 µM AA group compared with that in the raw semen (P<0.05). These findings suggest that supplementation of vitamin C to sperm medium could be beneficial for direct swim-up-derived spermatozoa.


Assuntos
Ácido Ascórbico/farmacologia , Separação Celular/métodos , Espermatozoides/fisiologia , Reação Acrossômica , Adulto , Ácido Ascórbico/administração & dosagem , Cromatina/patologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Relação Dose-Resposta a Droga , Glucose/farmacocinética , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Sêmen/fisiologia , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
9.
PLoS One ; 14(2): e0211837, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742661

RESUMO

Growing evidence suggests that paternal obesity may decrease male fertility potential. During infertility treatment with intra-cytoplasmic sperm injection (ICSI), a morphologically normal motile spermatozoon is injected into a mature egg, when possible. However, sperm motility and morphology per se do not reflect the sperm molecular composition. In this study, we aimed to assess the quality of motile spermatozoa in the context of obesity by analysing their conventional and molecular characteristics as well as their ability to promote early embryonic development. A prospective study was conducted on 128 infertile men divided into three groups: 40 lean, 42 overweight, and 46 obese men. Conventional sperm parameters (concentration, motility and morphology) and sperm molecular status (chromatin composition and integrity, 5-methycytosine (5-mC) and 5-hydroxycytosine (5-hmC) contents and oxidative stress level) were analysed on raw semen and/or on motile spermatozoa selected by density gradient or swim-up techniques. Morphokinetic analysis of the embryos derived from ICSI was performed using the Embryoviewer software. Our results showed that the motile sperm-enriched fraction from obese men exhibited higher levels of retained histones (p<0.001), elevated percentage of altered chromatin integrity (p<0.001), and decreased contents of 5-hmC (p<0.001), and 5-mC (p<0.05) levels as compared to that from lean men. Importantly, there were no statistically significant correlations between these molecular parameters and the percentages of morphologically normal motile spermatozoa. Regarding embryo morphokinetics, the CC1 (p<0.05) and CC3 (p<0.05) embryonic cell cycles were significantly delayed in the cleavage embryos of the obese group as compared to the embryos of the lean group. Our data is of particular interest because, besides demonstrating the negative impacts of obesity on motile spermatozoa molecular composition, it also highlights the possible risk of disturbing early embryonic cell cycles kinetics in the context of paternal obesity.


Assuntos
Cromatina , Obesidade , Motilidade Espermática , Espermatozoides , Adulto , Cromatina/metabolismo , Cromatina/patologia , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia , Estudos Prospectivos , Injeções de Esperma Intracitoplásmicas , Espermatozoides/metabolismo , Espermatozoides/patologia
10.
Nucleic Acids Res ; 47(8): 4124-4135, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30783677

RESUMO

A proper DNA damage response (DDR) is essential to maintain genome integrity and prevent tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion and their repair is orchestrated by the ATM kinase. ATM is activated via the MRE11-RAD50-NBS1 (MRN) complex along with its autophosphorylation at S1981 and acetylation at K3106. Activated ATM rapidly phosphorylates a vast number of substrates in local chromatin, providing a scaffold for the assembly of higher-order complexes that can repair damaged DNA. While reversible ubiquitination has an important role in the DSB response, modification of the newly identified ubiquitin-like protein ubiquitin-fold modifier 1 and the function of UFMylation in the DDR is largely unknown. Here, we found that MRE11 is UFMylated on K282 and this UFMylation is required for the MRN complex formation under unperturbed conditions and DSB-induced optimal ATM activation, homologous recombination-mediated repair and genome integrity. A pathogenic mutation MRE11(G285C) identified in uterine endometrioid carcinoma exhibited a similar cellular phenotype as the UFMylation-defective mutant MRE11(K282R). Taken together, MRE11 UFMylation promotes ATM activation, DSB repair and genome stability, and potentially serves as a therapeutic target.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Proteína Homóloga a MRE11/genética , Processamento de Proteína Pós-Traducional , Proteínas/genética , Reparo de DNA por Recombinação , Células A549 , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Cromatina/patologia , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Proteína Homóloga a MRE11/antagonistas & inibidores , Proteína Homóloga a MRE11/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Ligação Proteica , Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ubiquitinação
11.
Nature ; 565(7741): 659-663, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30675059

RESUMO

Replicative crisis is a senescence-independent process that acts as a final barrier against oncogenic transformation by eliminating pre-cancerous cells with disrupted cell cycle checkpoints1. It functions as a potent tumour suppressor and culminates in extensive cell death. Cells rarely evade elimination and evolve towards malignancy, but the mechanisms that underlie cell death in crisis are not well understood. Here we show that macroautophagy has a dominant role in the death of fibroblasts and epithelial cells during crisis. Activation of autophagy is critical for cell death, as its suppression promoted bypass of crisis, continued proliferation and accumulation of genome instability. Telomere dysfunction specifically triggers autophagy, implicating a telomere-driven autophagy pathway that is not induced by intrachromosomal breaks. Telomeric DNA damage generates cytosolic DNA species with fragile nuclear envelopes that undergo spontaneous disruption. The cytosolic chromatin fragments activate the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway and engage the autophagy machinery. Our data suggest that autophagy is an integral component of the tumour suppressive crisis mechanism and that loss of autophagy function is required for the initiation of cancer.


Assuntos
Autofagia , Carcinogênese/genética , Carcinogênese/patologia , Proliferação de Células , Instabilidade Cromossômica , Autofagia/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , Instabilidade Cromossômica/genética , Dano ao DNA/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Proteínas de Membrana/metabolismo , Membrana Nuclear/patologia , Nucleotidiltransferases/metabolismo , Telômero/genética , Telômero/patologia
12.
J Clin Invest ; 128(10): 4454-4471, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30179226

RESUMO

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.


Assuntos
Cromatina/metabolismo , Relógios Circadianos/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Glucocorticoides/efeitos adversos , Fígado/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Animais , Cromatina/genética , Cromatina/patologia , Relógios Circadianos/genética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Glucocorticoides/farmacologia , Células HEK293 , Humanos , Fígado/patologia , Camundongos , Camundongos Knockout , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo
13.
J Assist Reprod Genet ; 35(10): 1911-1917, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30056595

RESUMO

OBJECTIVE: To design a reproductive treatment algorithm based on the sperm DNA fragmentation (SDF) for couples with unexplained infertility following a poor intrauterine insemination (IUI) outcome. DESIGN: Couples that failed IUI with no apparent reproductive issue in both partners were allocated to diverse reproductive treatments on the basis of SDF. SETTING: Reproductive medical center in an academic setting. PATIENT(S): Over 4 years, couples with an unexpected poor IUI outcome and no apparent female or male partner reproductive issues were recruited. INTERVENTION(S): IUI, IVF, and ICSI were performed in the standard fashion following sperm SDF assays. MAIN OUTCOMES MEASURE(S): Fertilization rate, implantation rate, pregnancy characteristics, and delivery rates. RESULT(S): A total of 354 couples with unexplained infertility and normal semen parameters underwent 1133 IUI cycles. Clinical pregnancy rate (CPR) with IUI at our center in an age-matched cohort is 23.9% while the study cohort had 1.8%. Following SDF assessment, couples with failed IUI attempts but normal SDF (SCSA 9.8 ± 4.6%; TUNEL 11.8 ± 6.2%) underwent IVF with a CPR of 12.7%; those with abnormal SDF underwent ICSI with ejaculated spermatozoa, resulting in a CPR of 18.7%. This group included couples with normal SDF that had failed IVF. Couples with abnormal SDF that failed ICSI with ejaculated spermatozoa achieved a CPR of 31.0% with surgically retrieved spermatozoa. CONCLUSION(S): Couples with unexplained infertility that present with unexpectedly poor IUI outcomes can be funneled into a treatment algorithm guided by the integrity of the sperm genome for higher chances of pregnancy using an alternate method of insemination.


Assuntos
Cromatina/genética , Infertilidade Masculina/terapia , Sêmen , Espermatozoides/patologia , Adulto , Cromatina/patologia , Fragmentação do DNA , Feminino , Fertilização In Vitro , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Gravidez , Taxa de Gravidez , Análise do Sêmen , Contagem de Espermatozoides , Injeções de Esperma Intracitoplásmicas , Recuperação Espermática , Resultado do Tratamento
14.
J Clin Invest ; 128(8): 3605-3618, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29870403

RESUMO

While the genomic binding of MYC protein correlates with active epigenetic marks on chromatin, it remains largely unclear how major epigenetic mechanisms functionally impact the tumorigenic potential of MYC. Here, we show that, compared with the catalytic subunits, the core subunits, including DPY30, of the major H3K4 methyltransferase complexes were frequently amplified in human cancers and selectively upregulated in Burkitt lymphoma. We show that DPY30 promoted the expression of endogenous MYC and was also functionally important for efficient binding of MYC to its genomic targets by regulating chromatin accessibility. Dpy30 heterozygosity did not affect normal animal physiology including lifespan, but significantly suppressed Myc-driven lymphomagenesis, as cells failed to combat oncogene-triggered apoptosis as a result of insufficient epigenetic modulation and expression of a subset of antiapoptotic genes. Dpy30 reduction also greatly impeded MYC-dependent cellular transformation, without affecting normal cell growth. These results suggest that MYC hijacks a major epigenetic pathway - H3K4 methylation - to facilitate its molecular activity in target binding and to coordinate its oncogenic program for efficient tumorigenesis, meanwhile creating "epigenetic vulnerability." DPY30 and the H3K4 methylation pathway are thus potential epigenetic targets for treating certain MYC-driven cancers.


Assuntos
Linfoma de Burkitt/metabolismo , Transformação Celular Neoplásica/metabolismo , Cromatina/metabolismo , Epigênese Genética , Histona-Lisina N-Metiltransferase/biossíntese , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cromatina/genética , Cromatina/patologia , Histona-Lisina N-Metiltransferase/genética , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética
15.
J Cell Biochem ; 119(9): 7127-7142, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29923622

RESUMO

Nuclear alterations are a hallmark of many types of cancers, including prostate cancer (PCa). Recent evidence shows that subvisual changes, ones that may not be visually perceptible to a pathologist, to the nucleus and its ultrastructural components can precede visual histopathological recognition of cancer. Alterations to nuclear features, such as nuclear size and shape, texture, and spatial architecture, reflect the complex molecular-level changes that occur during oncogenesis. Quantitative nuclear morphometry, a field that uses computational approaches to identify and quantify malignancy-induced nuclear changes, can enable a detailed and objective analysis of the PCa cell nucleus. Recent advances in machine learning-based approaches can now automatically mine data related to these changes to aid in the diagnosis, decision making, and prediction of PCa prognoses. In this review, we use PCa as a case study to connect the molecular-level mechanisms that underlie these nuclear changes to the machine learning computational approaches, bridging the gap between the clinical and computational understanding of PCa. First, we will discuss recent developments to our understanding of the molecular events that drive nuclear alterations in the context of PCa: the role of the nuclear matrix and lamina in size and shape changes, the role of 3-dimensional chromatin organization and epigenetic modifications in textural changes, and the role of the tumor microenvironment in altering nuclear spatial topology. We will then discuss the advances in the applications of machine learning algorithms to automatically segment nuclei in prostate histopathological images, extract nuclear features to aid in diagnostic decision making, and predict potential outcomes, such as biochemical recurrence and survival. Finally, we will discuss the challenges and opportunities associated with translation of the quantitative nuclear morphometry methodology into the clinical space. Ultimately, accurate identification and quantification of nuclear alterations can contribute to the field of nucleomics and has applications for computationally driven precision oncologic patient care.


Assuntos
Cromatina/patologia , Interpretação de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Neoplasias da Próstata/patologia , Forma do Núcleo Celular , Tamanho do Núcleo Celular , Transformação Celular Neoplásica/ultraestrutura , Cromatina/ultraestrutura , Epigênese Genética , Instabilidade Genômica , Humanos , Masculino , Prognóstico , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/ultraestrutura , Microambiente Tumoral
16.
Trends Cancer ; 4(4): 320-331, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29606315

RESUMO

Abnormalities in nuclear and chromatin organization are hallmarks of many diseases including cancer. In this review, we highlight our understanding of how the cellular microenvironment regulates nuclear morphology and, with it, the spatial organization of chromosomes and genes, resulting in cell type-specific genomic programs. We also discuss the molecular basis for maintaining nuclear and genomic integrity and how alterations in nuclear mechanotransduction pathways result in various diseases. Finally, we highlight the importance of digital pathology based on nuclear morphometric features combined with single-cell genomics for early cancer diagnostics.


Assuntos
Núcleo Celular/patologia , Cromatina/patologia , Mecanotransdução Celular/genética , Neoplasias/diagnóstico , Microambiente Tumoral/genética , Biomarcadores Tumorais/análise , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Imagem Molecular/métodos , Mutação , Neoplasias/genética , Neoplasias/patologia , Progéria/diagnóstico , Progéria/genética , Progéria/patologia , Análise de Célula Única/métodos , Translocação Genética
17.
Andrology ; 6(3): 436-445, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29499098

RESUMO

The purpose of this study was to investigate the relationship between the proportion of sperm chromatin linked to remaining histone and assisted reproductive technology (ART) outcome. A prospective cohort study was performed on couples undergoing ART process at the Department of Reproduction Medicine (HFME, Bron, France). The histone-to-protamine ratio (HPR) was measured using the method described by Wykes & Krawetz (2003) J Biol Chem 278, 29471. The correlations with sperm DFI, blastocyst formation, pregnancy rate, and delivery rate were investigated. A total of 291 ART cycles were included (42 c-IVF and 249 ICSI procedures): 3870 oocytes were punctured and 2211 embryos were obtained, among which 507 were transferred and 336 frozen. The mean HPR was 18.9%. A significant negative correlation was found between HPR and DFI (r = -0.12, p < 0.05). Regarding the type of ART procedure (c-IVF or ICSI), the same kind of relationship between HPR and ART parameters was observed. Regardless of the type of ART procedure used, when the HPR was within the range [6%; 26%], the blastocyst formation rate was higher: 87.8% vs. 71.2% (HPR<6%; p < 0.01) and 74.6% (HPR >26%; p < 0.01). The highest delivery rate (DR; 24.5%) was obtained for HPR within the range [6%; 26%]; DR was 21.9% for HPR<6% and 18.3% for HPR>26%; however, the differences were not statistically significant. The procedure described in this study seems to be a reliable evaluation of the HPR. The HPR parameter seems to be correlated to embryonic development up to the blastocyst stage, but its involvement in clinical pregnancy/delivery could not be confirmed. HPR should be further investigated for confirming the relationship with blastocyst formation. After this, the next step will be to investigate the etiologies of HPR alterations for improving the sperm nucleus quality for increasing the chance of pregnancy.


Assuntos
Cromatina , Desenvolvimento Embrionário , Histonas , Protaminas , Técnicas de Reprodução Assistida , Espermatozoides , Adulto , Cromatina/metabolismo , Cromatina/patologia , Estudos de Coortes , Feminino , Histonas/metabolismo , Humanos , Masculino , Gravidez , Taxa de Gravidez , Estudos Prospectivos , Protaminas/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologia
18.
Mol Reprod Dev ; 85(4): 325-335, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29411926

RESUMO

Cryptochrome (Cry)1 is essential for generating circadian rhythm in central and many peripheral oscillators; however, its role in male reproduction remains unclear. We investigated this question using Cry1 knockout (KO) mice. We found that Cry1 is necessary for normal testicular function: Cry1 deficiency increased testicular germ cell apoptosis and decreased sperm count. A transcriptome analysis showed that the expression levels of 375 genes-including 12 encoding micro (mi)RNAs-were altered in the testis of Cry1 KO mice relative to wild-type controls. A bioinformatics analysis revealed that the differentially expressed genes were related to important biological processes including cell-cell communication, metabolism, chromatin reorganization, spermatogenesis, and the immune response. An integrative analysis of miRNA-mRNA networks suggested that the 12 dysregulated miRNAs may contribute to testis disorders through negative regulation of their target mRNAs expression in testis, and interestingly, all the 12 miRNAs are predicted to target core circadian clock components. These results provide the first evidence of a correlation between dysregulation of Cry1 and male reproductive defects in mice, indicating that Cry1 plays a critical role in maintaining normal testicular function.


Assuntos
Comunicação Celular , Cromatina/metabolismo , Criptocromos/deficiência , Regulação da Expressão Gênica , Espermatogênese , Testículo/metabolismo , Animais , Cromatina/patologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , MicroRNAs/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Testículo/patologia
19.
Diagn Cytopathol ; 46(5): 384-389, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29464913

RESUMO

OBJECTIVES: Development of computerized image analysis techniques has opened up the possibility for the quantitative analysis of nuclear chromatin in pathology. We hypothesized that the features extracted from digital images could be used to determine specific cytomorphological findings for nuclear chromatin that may be applicable for establishing a medical diagnosis. METHODS: Three parameters were evaluated from nuclear chromatin images obtained from the liquid-based cervical cytology samples of patients with biopsy-proven high-grade squamous intraepithelial lesion (HGSIL), and compared between non-neoplastic squamous epithelia and dysplastic epithelia groups: (1) standard deviation (SD) of the grayscale intensity; (2) difference between the maximum and minimum grayscale intensity (M-M); and (3) thresholded area percentage. Each parameter was evaluated at the mean, mean-1SD, and mean-2SD thresholding intensity levels. RESULTS: Between the mean and mean-1SD levels, the thresholded nuclear chromatin pattern was most similar to the chromatin granularity of the unthresholded grayscale images. The SD of the gray intensity and the thresholded area percentage differed significantly between the non-neoplastic squamous epithelia and dysplastic epithelia of HGSIL images at all three thresholding intensity levels (mean, mean-1SD, and mean-2SD). However, the M-M significantly differed between the two sample types for only two of the thresholding intensity levels (mean-1SD and mean-2SD). CONCLUSIONS: The digital parameters SD and M-M of the grayscale intensity, along with the thresholded area percentage could be useful in automated cytological evaluations. Further studies are needed to identify more valuable parameters for clinical application.


Assuntos
Cromatina/patologia , Interpretação de Imagem Assistida por Computador/métodos , Lesões Intraepiteliais Escamosas Cervicais/patologia , Núcleo Celular/patologia , Colo do Útero/patologia , Citodiagnóstico/métodos , Feminino , Humanos
20.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29321313

RESUMO

Hepatitis B virus (HBV) is a global major health problem, with over one million deaths annually caused by chronic liver damage. Understanding host factors that modulate HBV replication may aid the development of anti-HBV therapies. Our recent genome-wide small interfering RNA screen using recombinant HBV demonstrated that TIP60 inhibited HBV infection. Here, we show that TIP60 complex contributes to anti-HBV defense. The TIP60 complex bound to the HBV promoter and suppressed HBV transcription driven by the precore/core promoter. The silencing of EP400, TRRAP, BAF53a, RUVBL1, and RUVBL2, which form the TIP60 complex, also resulted in increased HBV transcription. These results contribute to our enhanced understanding of the molecular mechanism of HBV transcription associated with the chromatin structure of HBV covalently closed circular DNA (cccDNA). Exploiting these intrinsic cellular defenses might help develop new anti-HBV agents.IMPORTANCE Investigating the molecular mechanism of HBV replication is important to understand the persistent nature of HBV infection and to aid the development of new HBV agents, which are currently limited to HBV polymerase inhibitors. Previously, we developed a new reporter HBV. By screening host factors using this recombinant virus, we identified several gene products that regulate HBV infection, including TIP60. Here, we showed that TIP60, a catalytic subunit of the NuA4 complex, inhibited HBV replication. Depletion of TIP60 increased the level of HBV mRNA. Moreover, TIP60 localized in the HBV cccDNA chromatin complex catalyzed the acetylation of histone H4 to recruit Brd4. These results suggest that TIP60, in concert with other cellular factors, plays an important role in the regulation of the HBV chromatin structure by acting as a critical component of the intrinsic antiviral defense, which sheds new light on the regulation of HBV replication.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Lisina Acetiltransferase 5/metabolismo , Complexos Multiproteicos/metabolismo , Transcrição Genética , Replicação Viral/fisiologia , Acetilação , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , Cromatina/virologia , Células Hep G2 , Hepatite B/genética , Hepatite B/patologia , Histonas/genética , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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