RESUMEN
The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.
Asunto(s)
Búfalos , Proteómica , Animales , Femenino , Embarazo , Antioxidantes/metabolismo , Búfalos/metabolismo , Endometrio/metabolismo , Secretoma , Útero/metabolismoRESUMEN
Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with ß-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation.
Asunto(s)
Regulación de la Expresión Génica , ARN Polimerasa II , Humanos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Diferenciación Celular , Ciclo Celular , Transcripción Genética , Proteínas Nucleares/metabolismo , Factores de Elongación Transcripcional/genéticaRESUMEN
BACKGROUND AND OBJECTIVE: Outside clinical trials, there is limited long-term data following bronchial thermoplasty (BT). In a cohort of real-world severe asthmatics in an era of biological therapy, we sought to evaluate the safety and efficacy of BT 5 years post-treatment. METHODS: Every patient treated with BT at two Australian tertiary centres were recalled at 5 years, and evaluated by interview and record review, Asthma Control Questionnaire (ACQ), spirometry and high-resolution CT Chest. CT scans were interpreted using the modified Reiff and BRICS CT scoring systems for bronchiectasis. RESULTS: Fifty-one patients were evaluated. At baseline, this cohort had a mean age of 59.0 ± 11.8 years, mean ACQ of 3.0 ± 1.0, mean FEV1 of 55.5 ± 18.8% predicted, and 53% were receiving maintenance oral steroids in addition to triple inhaler therapy. At 5 years, there was a sustained improvement in ACQ scores to 1.8 ± 1.0 (p < 0.001). Steroid requiring exacerbation frequency was reduced from 3.8 ± 3.6 to 1.0 ± 1.6 exacerbations per annum (p < 0.001). 44% of patients had been weaned off oral steroids. No change in spirometry was observed. CT scanning identified minor degrees of localized radiological bronchiectasis in 23/47 patients with the modified Reiff score increasing from 0.6 ± 2.6 at baseline to 1.3 ± 2.5 (p < 0.001). However, no patients exhibited clinical features of bronchiectasis, such as recurrent bacterial infection. CONCLUSION: Sustained clinical benefit from BT at 5 years was demonstrated in this cohort of very severe asthmatics. Mild, localized radiological bronchiectasis was identified in a portion of patients without clinical features of bronchiectasis.
Asunto(s)
Asma , Termoplastia Bronquial , Bronquiectasia , Humanos , Persona de Mediana Edad , Anciano , Termoplastia Bronquial/efectos adversos , Termoplastia Bronquial/métodos , Corticoesteroides/uso terapéutico , Australia , Asma/tratamiento farmacológico , Bronquiectasia/diagnóstico por imagen , Bronquiectasia/cirugía , Bronquiectasia/tratamiento farmacológico , Esteroides/uso terapéuticoRESUMEN
The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with ß-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.
RESUMEN
The chromatin remodeller ATRX interacts with the histone chaperone DAXX to deposit the histone variant H3.3 at sites of nucleosome turnover. ATRX is known to bind repetitive, heterochromatic regions of the genome including telomeres, ribosomal DNA and pericentric repeats, many of which are putative G-quadruplex forming sequences (PQS). At these sites ATRX plays an ancillary role in a wide range of nuclear processes facilitating replication, chromatin modification and transcription. Here, using an improved protocol for chromatin immunoprecipitation, we show that ATRX also binds active regulatory elements in euchromatin. Mutations in ATRX lead to perturbation of gene expression associated with a reduction in chromatin accessibility, histone modification, transcription factor binding and deposition of H3.3 at the sequences to which it normally binds. In erythroid cells where downregulation of α-globin expression is a hallmark of ATR-X syndrome, perturbation of chromatin accessibility and gene expression occurs in only a subset of cells. The stochastic nature of this process suggests that ATRX acts as a general facilitator of cell specific transcriptional and epigenetic programmes, both in heterochromatin and euchromatin.
Asunto(s)
Cromatina , Heterocromatina , ADN Helicasas/genética , ADN Helicasas/metabolismo , Eucromatina/genética , Heterocromatina/genética , Histonas/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Nuclear Ligada al Cromosoma X/genética , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Talasemia alfaRESUMEN
The management of IIH during pregnancy is a topic of clinical importance and it may pose a management challenge as most cases of IIH occur in women of childbearing age. Although there is a consensus that pregnant women with IIH should be treated similarly to non-pregnant patients, there are uncertainties regarding optimal management. This review aims to analyse current evidence and literature to help guide management of IIH during pregnancy. It is recommended that pregnant women with IIH are treated in health care settings that have access to multi-specialty input to optimise treatment. The management depends on disease severity with a treatment paradigm that encompasses conservative, medical and surgical management.
Asunto(s)
Hipertensión Intracraneal , Seudotumor Cerebral , Femenino , Humanos , Hipertensión Intracraneal/diagnóstico , Hipertensión Intracraneal/terapia , Embarazo , Seudotumor Cerebral/diagnóstico , Seudotumor Cerebral/terapia , Índice de Severidad de la EnfermedadRESUMEN
This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.
Asunto(s)
Acrosina , Proteómica , Acrosina/metabolismo , Animales , Perros , Masculino , Fitomejoramiento , Proteómica/métodos , Semen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Albúmina Sérica/metabolismo , Motilidad Espermática , Espermatozoides/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
OBJECTIVE: The safety of stereo-electroencephalography (SEEG) has been investigated; however, most studies have not differentiated pediatric and adult populations, which have different anatomy and physiology. The purpose of this study was to assess SEEG safety in the pediatric setting, focusing on surgical complications and the identification of patient and surgical risk factors, if any. The authors also aimed to determine whether robot assistance in SEEG was associated with a change in practice, surgical parameters, and clinical outcomes. METHODS: The authors retrospectively studied all SEEG cases performed in their department from December 2014 to March 2020. They analyzed both demographic and surgical variables and noted the types of surgery-related complications and their management. They also studied the clinical outcomes of a subset of the patients in relation to robot-assisted and non-robot-assisted SEEG. RESULTS: Sixty-three children had undergone 64 SEEG procedures. Girls were on average 3 years younger than the boys (mean age 11.1 vs 14.1 years, p < 0.01). The overall complication rate was 6.3%, and the complication rate for patients with left-sided electrodes was higher than that for patients with right-sided electrodes (11.1% vs 3.3%), although the difference between the two groups was not statistically significant. The duration of recording was positively correlated to the number of implanted electrodes (r = 0.296, p < 0.05). Robot assistance was associated with a higher number of implanted electrodes (mean 12.6 vs 7.6 electrodes, p < 0.0001). Robot-assisted implantations were more accurate, with a mean error of 1.51 mm at the target compared to 2.98 mm in nonrobot implantations (p < 0.001). Clinical outcomes were assessed in the first 32 patients treated (16 in the nonrobot group and 16 in the robot group), 23 of whom proceeded to further resective surgery. The children who had undergone robot-assisted SEEG had better eventual seizure control following subsequent epilepsy surgery. Of the children who had undergone resective epilepsy surgery, 42% (5/12) in the nonrobot group and 82% (9/11) in the robot group obtained an Engel class IA outcome at 1 year (χ2 = 3.885, p = 0.049). Based on Kaplan-Meier survival analysis, the robot group had a higher seizure-free rate than the nonrobot group at 30 months postoperation (7/11 vs 2/12, p = 0.063). Two complications, whose causes were attributed to the implantation and head-bandaging steps, required surgical intervention. All complications were either transient or reversible. CONCLUSIONS: This is the largest single-center, exclusively pediatric SEEG series that includes robot assistance so far. SEEG complications are uncommon and usually transient or treatable. Robot assistance enabled implantation of more electrodes and improved epilepsy surgery outcomes, as compared to those in the non-robot-assisted cases.
Asunto(s)
Epilepsia Refractaria/cirugía , Electroencefalografía/métodos , Procedimientos Neuroquirúrgicos/instrumentación , Procedimientos Neuroquirúrgicos/métodos , Procedimientos Quirúrgicos Robotizados/instrumentación , Procedimientos Quirúrgicos Robotizados/métodos , Convulsiones/cirugía , Adolescente , Niño , Preescolar , Epilepsia Refractaria/diagnóstico por imagen , Electrodos Implantados , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Procedimientos Neuroquirúrgicos/efectos adversos , Complicaciones Posoperatorias/epidemiología , Complicaciones Posoperatorias/cirugía , Estudios Retrospectivos , Procedimientos Quirúrgicos Robotizados/efectos adversos , Convulsiones/diagnóstico por imagen , Técnicas Estereotáxicas , Análisis de Supervivencia , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
ObjectiveTo explore end-of-life care in the ward and intensive care unit (ICU) environment in nine Australian hospitals in a retrospective observational study.MethodsIn total, 1693 in-hospital deaths, 356 in ICU, were reviewed, including patient demographics, advance care plans, life-sustaining treatments, recognition of dying by clinicians and evidence of the palliative approach to patient care.ResultsMost patients (n=1430, 84%) were aged ≥60 years, with a low percentage (n=208, 12%) having an end-of-life care plan on admission. Following admission, 82% (n=1391) of patients were recognised as dying, but the time between recognition of dying to death was short (ICU (staying 4-48h) median 0.34 days (first quartile (Q1), third quartile (Q3): 0.16, 0.72); Ward (staying more than 48h) median 2.1 days (Q1, Q3: 0.96, 4.3)). Although 41% (n=621) patients were referred for specialist palliative care, most referrals were within the last few days of life (2.3 days (0.88, 5.9)) and 62% of patients (n=1047) experienced active intervention in their final 48h.ConclusionsLate recognition of dying can expose patients to active interventions and minimises timely palliative care. To attain alignment to the National Consensus Statement to improve experiences of end-of-life care, a nationally coordinated approach is needed.What is known about the topic?The majority of Australian patient deaths occur in hospitals whose care needs to align to the Australian Commission on Safety and Quality in Health Care's National Consensus Statement, essential elements of safe and high-quality end-of-life care.What does this paper add?The largest Australian study of hospital deaths reveals only 12% of patients have existing advance care plans, recognition of death is predominantly within the last 48h of life, with 60% receiving investigations and interventions during this time with late symptom relief.What are the implications for practitioners?Given the poor alignment with the National Consensus Statement, a nationally coordinated approach would improve the patient experience of end-of-life care.
RESUMEN
BACKGROUND: Congenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate. METHODS: Genetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation. RESULTS: We identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells. CONCLUSION: Stability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Predisposición Genética a la Enfermedad , Glicoproteínas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Anemia Diseritropoyética Congénita/patología , Femenino , Regulación de la Expresión Génica/genética , Pruebas Genéticas , Genética de Población , Humanos , Masculino , Complejos Multiproteicos/genética , Mutación/genéticaRESUMEN
The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic 'spongy' heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.
Asunto(s)
Anemia Diseritropoyética Congénita , Anemia Diseritropoyética Congénita/diagnóstico , Anemia Diseritropoyética Congénita/genética , Células Eritroides , Eritropoyesis , Glicoproteínas/genética , Humanos , Proteínas Nucleares/genéticaRESUMEN
Semen contains several proteins that are important to fertilization and to identify reproductive failures. There are proteins that are specie-specific expressed, although differs among several breeds. This article provides experimental data describing the protein profile of seminal plasma and spermatozoa of four healthy purebred dogs: Golden Retriever (n=3), Bernese Mountain Dog (n=4), Great Dane (n=3), and Maremmano-Abruzzese Sheepdog (n=3), housed at São Paulo state, Brazil. Semen samples were collected by manual stimulation of the penis in a presence of a teaser bitch, when possible. The seminal plasma and sperm cells were separated by centrifugation and prepared for mass spectrometry. The gene ontology annotation of the proteins found is described. This is the first time that proteomic profile of the semen of purebred dogs is described. These data are a valuable resource to improve the biotechnologies of reproduction applied to canid species.
RESUMEN
BACKGROUND: In the context of a variable condition such as asthma, patient recognition of deteriorating control and knowing what prompt action to take is crucial. Yet, implementation of recommended self-management strategies remains poor. AIM: To explore how patients with asthma and parents/carers of children with asthma develop and establish recommended self-management strategies for living with asthma, and how clinicians can best support the process. DESIGN AND SETTING: A qualitative study in UK primary care. METHOD: Patients with asthma and parents/carers of children with asthma from 10 general practices were purposively sampled (using age, sex, and duration of asthma) to participate in focus groups or interviews between May 2016 and August 2016. Participants' experiences of health care, management of asthma, and views on supported self-management were explored. Interviews and focus group sessions were audio-recorded and transcribed verbatim. Iterative thematic analysis was conducted, guided by the research questions and drawing on habit theory in discussion with a multidisciplinary research team. RESULTS: A total of 49 participants (45 patients; 4 parents/carers) took part in 32 interviews and five focus groups. Of these, 11 reported using an action plan. Patients learnt how to self-manage over time, building knowledge from personal experience and other sources, such as the internet. Some regular actions, for example, taking medication, became habitual. Dealing with new or unexpected scenarios required reflective abilities, which may be supported by a tailored action plan. CONCLUSION: Patients reported learning intuitively how to self-manage. Some regular actions became habitual; dealing with the unexpected required more reflective cognitive skills. In order to support implementation of optimal asthma self- management, clinicians should consider both these aspects of self-management and support, and educate patients proactively.
Asunto(s)
Asma , Niño , Humanos , Asma/terapia , Atención Primaria de Salud , Investigación Cualitativa , Reino UnidoRESUMEN
Sexual and gender minority (SGM) medical students and physicians are exposed to bias in professional contexts. One strategy for promoting SGM visibility and inclusion within medicine is the development of institutional OutLists, which are online, opt-in lists of SGM-identified individuals affiliated with an academic institution. We present the first quantitative evaluation of publicly accessible OutLists at medical institutions in the United States, Canada, and Europe. Nineteen OutLists were identified in the United States; no OutLists were identified in other countries. All OutLists in the United States were identified at allopathic institutions with no institutional religious affiliation. Clinicians in high-prestige specialties and more senior clinicians were underrepresented on OutLists. A state-level measure of SGM equality predicted presence of OutLists within the state (odds ratio 1.429, p = .047) but was not associated with the total number of individuals on OutLists. Future research would benefit from incorporating qualitative methodologies to explore the effectiveness of OutLists and the individual experiences of participants in these lists.
Asunto(s)
Instituciones de Salud/estadística & datos numéricos , Homosexualidad , Autorrevelación , Minorías Sexuales y de Género , Canadá , Humanos , Médicos , Estudiantes de Medicina , Estados UnidosRESUMEN
Healthcare professionals have limited formal end-of-life care training despite the large proportion of hospital deaths. A retrospective review of 201 acute hospital deaths revealed 166 (82.6%) had documentation to suggest the patient was dying but this was performed late with a median time between documentation and death of 0.84 days. Furthermore, 132 (66%) patients received an intervention in the final 48 h of life. This highlights the need to improve the recognition and management of dying patients in acute hospitals.
Asunto(s)
Competencia Clínica/normas , Toma de Decisiones , Documentación/estadística & datos numéricos , Cuidado Terminal/normas , Femenino , Humanos , Masculino , Rol del Médico , Vigilancia de Guardia , Factores de TiempoRESUMEN
Sexed sperm in dogs is of interest because of being polytocous, and as a result, the greatest number of offspring of the same sex can improve the market, although few studies assessing sperm sexing have been performed in this species. The present study, therefore, was conducted to evaluate the effects on sperm quality and the effectiveness of three discontinuous density gradients to separate dog sperm containing X and Y chromosomes. Thirty ejaculates from ten adult dogs were collected by digital manipulation of the penis. Cells were separated using gradients of Percoll® and Percoll® associated with Nycodenz® or Ficoll. The cells were evaluated for motility by the CASA system (Computer-Aided Semen Analyzer) and for concentration and recovered sperm concentration (after centrifugation), sperm morphology, plasma and acrosomal membrane integrity, and mitochondrial function pre- and post-centrifugation. The percentage of sperm containing X and Y chromosomes was also evaluated pre- and post-centrifugation by quantitative real-time PCR (qPCR). The use of the Ficoll gradient resulted in the greatest sperm quality after centrifugation; however, no sperm enhancement containing X or Y chromosome occurred with use of any of the methods (Percoll® 54.8 ± 1.9 compared with 45.2 ± 1.9; Percoll® associated with Nycodenz® 53.2 ± 2.0 compared with 46.8 ± 2.0; and Percoll® associated with Ficoll 55.0 ± 1.5 compared with 45.0 ± 1.5 for the percentages of cells containing the X and Y chromosomes, respectively). Thus, it was concluded that the technique of sexing dog sperm using density gradients was not effective for commercial application.
Asunto(s)
Centrifugación por Gradiente de Densidad/veterinaria , Perros , Preselección del Sexo/veterinaria , Espermatozoides/fisiología , Animales , Separación Celular/métodos , Masculino , Cromosomas Sexuales , Preselección del Sexo/métodosRESUMEN
The identification of distinct proteins present on the membrane of spermatozoa with X and Y chromosomes allows the development of immuno-sexing techniques. The aim of this study, therefore, was to use mass spectrometry to analyze the protein profile of sperm previously categorized using flow cytometry into X or Y-bearing semen pools. Sex-sorted sperm samples (n = 6 X and n = 6 Y) were used. Proteins were extracted and analyzed by mass spectrometry using data independent acquisition (DIA). The data were searched against taxonomy Bos taurus in the Swiss Prot database. In total, 459 protein groups were identified. Of these, eight proteins were in differential abundances between the X- and Y-bearing sperm population. Among the major proteinsdetected, EF-hand domain-containing protein 1, a protein involved in embryonic development, is more abundant in Y-bearing spermatozoa. In addition, proteins FUN14, domain-containing protein 2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 mitochondrial, cytochrome C oxidase subunit 2, acetyl -CoA carboxylase type beta were more abundant in X-bearing sperm. In conclusion, there were differences in abundance of proteins between X- and Y-bearing bull spermatozoa. This fact, may contribute to future studies related to sperm physiology and possibility development of immuno-sexing techniques.
Asunto(s)
Espectrometría de Masas/métodos , Proteoma/análisis , Preselección del Sexo , Espermatozoides/citología , Espermatozoides/metabolismo , Cromosoma X/metabolismo , Cromosoma Y/metabolismo , Animales , Bovinos , Separación Celular/métodos , Citometría de Flujo/métodos , Inmunoensayo , Masculino , Proteómica , Preselección del Sexo/veterinaria , Espermatozoides/químicaRESUMEN
Self-interacting chromatin domains encompass genes and their cis-regulatory elements; however, the three-dimensional form a domain takes, whether this relies on enhancer-promoter interactions, and the processes necessary to mediate the formation and maintenance of such domains, remain unclear. To examine these questions, here we use a combination of high-resolution chromosome conformation capture, a non-denaturing form of fluorescence in situ hybridisation and super-resolution imaging to study a 70 kb domain encompassing the mouse α-globin regulatory locus. We show that this region forms an erythroid-specific, decompacted, self-interacting domain, delimited by frequently apposed CTCF/cohesin binding sites early in terminal erythroid differentiation, and does not require transcriptional elongation for maintenance of the domain structure. Formation of this domain does not rely on interactions between the α-globin genes and their major enhancers, suggesting a transcription-independent mechanism for establishment of the domain. However, absence of the major enhancers does alter internal domain interactions. Formation of a loop domain therefore appears to be a mechanistic process that occurs irrespective of the specific interactions within.