A matter of wrapper: Defects in the nuclear envelope of lagging and bridging chromatin threatens genome integrity.

The nuclear envelope surrounds the eukaryotic genome and, through the nuclear pore complexes, regulates transport in and out of the nucleus. Correct nucleo-cytoplasm compartmentations are essential for nuclear functions such as DNA replication or repair. During metazoan mitosis, the nuclear envelope disintegrates to allow the segregation of the two copies of DNA between daughter cells. At the end of mitosis, it reforms on each group of chromosomes in the daughter cells. However, nuclear envelope reformation is delayed on lagging chromosomes and DNA bridges. Defects in the coordination between nuclear envelope reformation and chromosome segregation impair the nuclear functions. Mechanical stress to which micronuclei and DNA bridges are subjected to combined with their particular architecture and the altered nuclear functions result in DNA damage. While micronuclei and DNA bridges were considered for more than 100 years as mere indicators of chromosomal instability, rapid technological advances are helping to better understand the biological consequences of these aberrant nuclear morphologies. Recent studies provide interesting evidence that micronuclei and chromatin bridges act as a key platforms for a catastrophic mutational process observed in cancers called chromothripsis and a trigger for the innate immune response. Therefore, they could affect cellular functions by both genetic and non-genetic means.

Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells.

Chromosomal instability, the most frequent form of plasticity in cancer cells, often proceeds through the formation of chromosome bridges. Despite the importance of these bridges in tumor initiation and progression, debate remains over how and when they are resolved. In this study, we investigated the behavior and properties of chromosome bridges to gain insight into the potential mechanisms underlying bridge-induced genome instability. We report that bridges may break during mitosis or may remain unbroken until the next interphase. During mitosis, we frequently observed discontinuities in the bridging chromatin, and our results strongly suggest that a substantial fraction of chromosome bridges are broken during this stage of the cell cycle. This notion is supported by the observation that the chromatin flanking mitotic bridge discontinuities is often decorated with the phosphorylated form of the histone H2AX, a marker of DNA breaks, and by MDC1, an early mediator of the cell response to DNA breaks. Also, free 3'OH DNA ends were detected in more than half of the bridges during the final stages of cell division. However, even if detected, the DNA ends of broken bridges are not repaired in mitosis. To investigate whether mitotic bridge breakage depends on mechanical stress, we used experimental models in which chromosome bridges with defined geometry are formed. Although there was no association between spindle pole separation or the distance among non-bridge kinetochores and bridge breakage, we found a direct correlation between the distance between bridge kinetochores and bridge breakage. Altogether, we conclude that the discontinuities observed in bridges during mitosis frequently reflect a real breakage of the chromatin and that the mechanisms responsible for chromosome bridge breakage during mitosis may depend on the separation between the bridge kinetochores. Considering that previous studies identified mechanical stress or biochemical digestion as possible causes of bridge breakage in interphase cells, a multifactorial model emerges for the breakage of chromosome bridges that, according to our results, can occur at different stages of the cell cycle and can obey different mechanisms.

Age-associated deficient recruitment of 53BP1 in G1 cells directs DNA double-strand break repair to BRCA1/CtIP-mediated DNA-end resection.

DNA repair mechanisms play a crucial role in maintaining genome integrity. However, the increased frequency of DNA double-strand breaks (DSBs) and genome rearrangements in aged individuals suggests an age-associated DNA repair deficiency. Previous work from our group revealed a delayed firing of the DNA damage response in human mammary epithelial cells (HMECs) from aged donors. We now report a decreased activity of the main DSB repair pathways, the canonical non-homologous end-joining (c-NHEJ) and the homologous recombination (HR) in these HMECs from older individuals. We describe here a deficient recruitment of 53BP1 to DSB sites in G1 cells, probably influenced by an altered epigenetic regulation. 53BP1 absence at some DSBs is responsible for the age-associated DNA repair defect, as it permits the ectopic formation of BRCA1 foci while still in the G1 phase. CtIP and RPA foci are also formed in G1 cells from aged donors, but RAD51 is not recruited, thus indicating that extensive DNA-end resection occurs in these breaks although HR is not triggered. These results suggest an age-associated switch of DSB repair from canonical to highly mutagenic alternative mechanisms that promote the formation of genome rearrangements, a source of genome instability that might contribute to the aging process.

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells.

Tumorigenesis is a multi-step process in which cells acquire capabilities that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions. A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay. Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

Identification of reference genes for RT-qPCR data normalisation in aging studies.

Aging is associated with changes in gene expression levels that affect cellular functions and predispose to age-related diseases. The use of candidate genes whose expression remains stable during aging is required to correctly address the age-associated variations in expression levels. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR) has become a powerful approach for sensitive gene expression analysis. Reliable RT-qPCR assays rely on the normalisation of the results to stable reference genes. Taken these data together, here we evaluated the expression stability of eight frequently used reference genes in three aging models: oncogene-induced senescence (OIS), in vitro and in vivo aging. Using NormFinder and geNorm algorithms, we identified that the most stable reference gene pairs were PUM1 and TBP in OIS, GUSB and PUM1 for in vitro aging and GUSB and OAZ1 for in vivo aging. To validate these candidates, we used them to normalise the expression data of CDKN1A, APOD and TFRC genes, whose expression is known to be affected during OIS, in vitro and in vivo aging. This study demonstrates that accurate normalisation of RT-qPCR data is crucial in aging research and provides a specific subset of stable reference genes for future aging studies.

Delayed γH2AX foci disappearance in mammary epithelial cells from aged women reveals an age-associated DNA repair defect.

Aging is a degenerative process in which genome instability plays a crucial role. To gain insight into the link between organismal aging and DNA repair capacity, we analyzed DNA double-strand break (DSB) resolution efficiency in human mammary epithelial cells from 12 healthy donors of young and old ages. The frequency of DSBs was measured by quantifying the number of γH2AX foci before and after 1Gy of γ-rays and it was higher in cells from aged donors (ADs) at all times analyzed. At 24 hours after irradiation, ADs retained a significantly higher frequency of residual DSBs than young donors (YDs), which had already reached values close to basal levels. The kinetics of DSB induction and disappearance showed that cells from ADs and YDs repair DSBs with similar speed, although analysis of early times after irradiation indicate that a repair defect may lie within the firing of the DNA repair machinery in AD cells. Indeed, using a mathematical model we calculated a constant factor of delay affecting aged human epithelial cells repair kinetics. This defect manifests with the accumulation of DSBs that might eventually undergo illegitimate repair, thus posing a relevant threat to the maintenance of genome integrity in older individuals.

Radiation-Induced Malignant Transformation of Preneoplastic and Normal Breast Primary Epithelial Cells.

Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. IMPLICATIONS: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.

A randomized comparison of the Ambu AuraGain versus the LMA supreme in patients undergoing gynaecologic laparoscopic surgery.

Second generation supraglottic airway devices providing high seal airway pressures are suitable for patients undergoing gynecologic laparoscopy. We compared the seal pressure achieved by the new Ambu AuraGain™ versus LMA Supreme™ following pneumoperitoneum in the Trendelenburg position. Sixty female patients were randomly allocated to ventilation with either the AuraGain or the Supreme. A target-controlled system was used to administer total intravenous anesthesia. Intracuff pressure was maintained below 60 cm H2O. The following parameters were registered: Time, number of attempts and manoeuvres required for insertion; seal pressure and peak inspiratory pressure at four time points; ease of gastric tube insertion, flexible scope view, complications and postoperative morbidity. Both devices were quick and easily inserted, although the Supreme required less rotation manoeuvres (16 in AuraGain vs. 6 in LMA Supreme; p = 0.01). The AuraGain achieved higher seal pressures (34 ± 5 in AuraGain vs. 29 ± 5 in LMA Supreme; p = 0.0002). Following pneumoperitoneum in head-down position, peak airway pressure increased 9 ± 3 cm H2O in both groups, exceeding seal pressure in 3 patients in the Supreme group (p = 0.06). The vocal cords were seen through all AuraGain and 90% of the Supreme devices; epiglottis was often visible inside the tube (68%). No differences were found in the incidence of traces of blood on the mask or postoperative symptoms. Both devices allowed effective ventilation in patients undergoing gynaecologic laparoscopic surgery with a low rate of complications. The Ambu AuraGain provided higher seal pressures and a clear view of glottic inlet in all patients offering the possibility to guide direct tracheal intubation if required.

Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis.

In order to examine the relationship between accumulation of residual DNA double-strand breaks (DSBs) and cell death, we have used a control and an ATM (Ataxia-Telangiectasia Mutated) defective cell line, as Ataxia-Telangiectasia (AT) cells tend to accumulate residual DSBs at long times after damage infliction. After irradiation, AT cells showed checkpoint impairment and a fraction of cells displayed an abnormal centrosome number and tetraploid DNA content, and this fraction increased along with apoptosis rates. At all times analyzed, AT cells displayed a significantly higher rate of radiation-induced apoptosis than normal cells. Besides apoptosis, 70-85% of the AT viable cells (TUNEL-negative) carried ≥ 10 γH2AX foci/cell, while only 12-27% of normal cells did. The fraction of AT and normal cells undergoing early and late apoptosis were isolated by flow cytometry and residual DSBs were concretely scored in these populations. Half of the γH2AX-positive AT cells undergoing early apoptosis carried ≥ 10 γH2AX foci/cell and this fraction increased to 75% in late apoptosis. The results suggest that retention of DNA damage-induced γH2AX foci is an indicative of lethal DNA damage, as cells undergoing apoptosis are those accumulating more DSBs. Scoring of residual γH2AX foci might function as a predictive tool to assess radiation-induced apoptosis.

Representaciones del cuerpo en el embarazo. Experiencias de las mujeres / The representations of the body during pregnancy. Women´s experiences

Objetivo: Este artículo tiene como objetivo analizar las representacionesque una serie de mujeres embarazadas expusieron sobre el cuerpo,revisando las influencias externas sobre sus experiencias.Diseño: Metodología cualitativa con un enfoque antropológico.Resultados: Las interpretaciones y experiencias del cuerpo fueron diversasy evolucionaron a lo largo del embarazo. Entre ellas destacamossu percepción positiva, al responder de forma natural y automática,aunque conllevara a su transformación-deformación. La previsión delaumento de peso fue una imagen preocupante para la mayoría y, aunqueaceptada, se tradujo en prácticas de autocontrol. Otras experienciasfueron de ocupación e interpretación del cuerpo a partir de los discursosprofesionales.Conclusiones: Conocer las representaciones que las mujeres hacen desus cuerpos en gestación y revisar las propias puede mejorar la efectividadde los cuidados (AU)

Objetive: The purpose of this paper is to analyze the representations ofthe body presented by pregnant women, through the review of their influenceon their experiences.Design: Qualitative methodology with a anthropologic design.Results: Their corporal perceptions and experiences were very diverseand developed throughout the pregnancy. Among them, we highlighttheir positive perception, due to the natural and automatic response,although it leads to its transformation-deformation. The predictableweight gain was a worrying image for most of them. The prediction ofweight gain was an image of concern for most of them, and, althoughthey accepted it, it leads to self-control practices. Other experienceswere of feelings of invasion and interpretation of their body followinginteractions with professionals.Conclusions: Knowing women’s representations of their pregnant bodiesand revising our own may improve the effectiveness of prenatal care (AU)

A clinical evaluation of four disposable laryngeal masks in adult patients.

STUDY OBJECTIVE: To compare the clinical use of four disposable laryngeal masks (DLMs): the Ambu laryngeal mask [Ambu LM], Solus, Laryngeal Mask Airway (LMA) Unique, and Soft Seal. DESIGN: Prospective, randomized study. SETTING: Operating room and recovery area of a university-affiliated ambulatory surgery unit. PATIENTS: 200 adult ASA physical status I, II, and III patients, scheduled for ambulatory procedures. INTERVENTIONS: Patients underwent insertion of the DLM by nonexperienced residents. MEASUREMENTS: The time and number of attempts needed for insertion, quality of ventilation, airway sealing pressure at 60 cmH(2)O of intracuff pressure, and complications were all evaluated. MAIN RESULTS: Ease of insertion was greater (P = 0.03) and first attempt success rate was higher with the Ambu LM (78%) and LMA Unique (80%). The Solus most often needed three attempts to be placed (12 cases); the Ambu LM needed three similar attempts in two cases; the LMA Unique in 4 cases; and the Soft Seal in 5 cases (P = 0.018). The LMA Unique achieved the highest rate of optimal ventilation (46/49 cases) of the 4 groups. Airway sealing pressure was significantly higher with the Soft Seal (27.3 +/- 5 mmHg), compared to the Ambu LM (23.7 +/- 5 mmHg), the Solus (20.9 +/- 4 mmHg), and the LMA Unique (22.1 +/- 6 mmHg) (P < 0.001). Blood staining of the DLM on removal was most frequent with the Soft Seal (38%). CONCLUSIONS: The Ambu LM and LMA Unique DLMs appear to be easier to insert by inexperienced residents and are less traumatic for the patient. The Soft Seal achieves a higher airway seal than other devices, but it causes more mucosal trauma. The Solus had the highest insertion failure rate of the 4 groups.

Hyperlactatemia in patients with non-acetaminophen-related acute liver failure.

AIM: To characterize hyperlactatemia in patients with non-acetaminophen acute liver failure (ALF) in an attempt to clarify the mechanisms implicated and the role as a prognosis factor. METHODS: In the setting of liver transplantation, 63 consecutive patients with non-acetaminophen acute liver failure were studied in relation to tissue oxygenation, hemodynamic and metabolic parameters. Before and after transplantation, the number of infected patients and outcome were registered. RESULTS: Acute ALF showed higher levels of lactate than subacute ALF (5.4+/- 1 mmol/L versus 2.2+/- 0.6 mmol/L, P=0.01). Oxygenation parameters were within the normal range. Lactate levels showed good correlation with respiratory quotient (r=0.759, P< 0.005), mean glucose administration (r=0.664, P=0.01) and encephalopathy (r=0.698, P=0.02), but not with splanchnic arteriovenous difference in PCO2, pH and the presence of infection (P=0.1). Portal vein lactate was higher (P< 0.05) than arterial and mixed venous lactate, suggesting its production of hyperlactatemia in the intestine and spleen. The presence of infection was an independent predictor of survival. CONCLUSION: Hyperlactatemia is not a prognosis factor due to byproduct of the overall acceleration in glycolysis.

Postoperative pain relief after hepatic resection in cirrhotic patients: the efficacy of a single small dose of ketamine plus morphine epidurally.

In cirrhotic patients undergoing hepatic surgery, postoperative analgesia remains a challenge. In this study, we evaluated the efficacy of a single dose of morphine combined with small-dose ketamine given epidurally for postoperative pain relief. One-hundred-four classification "Child A" cirrhotic patients were randomly assigned to two groups: 1) (MKG, n = 54): epidural morphine (3.5-5 mg) plus ketamine (20/30 mg); and 2) epidural morphine (3.5/5 mg) (MG, n = 50). The level of analgesia, side effects, psychomimetic and neurological disorders, additional analgesic needs, and overall quality of the analgesia were recorded. The mean duration of analgesia was longer in the MKG group (27.2 +/- 8 h versus 16.4 +/- 10 h; P < 0.05). In the MKG group, the visual analog scale (VAS) score began to be significantly lower from 14 h at rest and 12 h on coughing until the end of the study. The need for additional analgesia was also smaller in the MKG group (P < 0.05): at 24 h, only 10% of patients in the MKG group needed complementary analgesia, whereas in the MG group it was 100% (P = 0.003). Side effects were similar in both groups. Psychomimetic side effects and neurological disorders were not detected. These results suggest that postoperative analgesia provided by a single dose of epidural morphine with small-dose ketamine is effective in cirrhotic Child's A patients having major upper abdominal surgery.

Epidural somatostatin as an analgesic in upper abdominal surgery: a double-blind study.

Somatostatin 1-14, a natural occurring neuropeptide (Somiaton), has been reported to have analgesic effects in humans in different painful conditions. The aim of the present study was to investigate if epidural somatostatin produced clinical analgesia to postoperative pain after upper abdominal surgery. In a randomized double-blind controlled study, 40 patients received either 125 micrograms of epidural somatostatin infusions every hour (using a continuous infusion pump: CADD-PCA model 5200 PCX, Pharmacia) or placebo: mannitol (somatostatin inactif ingredient) 2.5 mg during the first 3 postoperative days (plus additional pulses of either substance, 250 micrograms or 5 mg, respectively, according to the level of analgesia needed by the patient). Additional subcutaneous analgesic treatment with 1 mg/kg pethidine chlorhydrate was administered at the patient's request. The degree of pain was quantified with visual analogue scale at baseline, 1 h after the operation and at every 4 h for the next 3 days. Arterial blood gases and spirometry values were determined at different intervals throughout the study period. Somatostatin was significantly better than placebo for pain relief (P < 0.01) and respiratory function preservation (P < 0.05). The total consumption (and ranges) of somatostatin at 24, 48 and 72 h were: 5.2 +/- 1.4 mg (4.0-6.25 mg), 4.2 +/- 0.8 mg (2.2-5.0 mg) and 3.7 +/- 0.4 mg (2.2-4.7 mg) respectively. During the whole study the need for complementary analgesia (pethidine chlorhydrate) was significantly higher in the placebo group: 5.4 +/- 3.5 vs. 2.7 +/- 1.9 (mean +/- SD) P < 0.01, dose/72 h. Side effects were irrelevant and scarce in both groups. The sustained pain relief combined with the respiratory function preservation in the somatostatin group suggests an important role of this drug in postoperative analgesia.