Broken chromosomes heading into mitosis: More than one way to patch a flat tire.

A cell dealing with a broken chromosome in mitosis is like a driver dealing with a flat tire on the highway: damage repair must occur under non-ideal circumstances. Mitotic chromosome breaks encounter problems related to structures called micronuclei. These aberrant nuclei are linked to cell death, mutagenesis, and cancer. In the last few years, a flurry of studies illuminated two mechanisms that prevent mitotic problems related to micronuclei. One mechanism prevents micronuclei from forming during mitosis and involves DNA Polymerase Theta, a DNA repair regulator that patches up broken mitotic chromosomes. A second mechanism is activated after micronuclei form and then rupture, and involves CIP2A and TOPBP1 proteins, which patch micronuclear fragments to promote their subsequent mitotic segregation. Here, we review recent progress in this field of mitotic DNA damage and discuss why multiple mechanisms exist. Future studies in this exciting area will reveal new DNA break responses and inform therapeutic strategies.

Rap1 promotes epithelial integrity and cell viability in a growing tissue.

Having intact epithelial tissues is critical for embryonic development and adult homeostasis. How epithelia respond to damaging insults or tissue growth while still maintaining intercellular connections and barrier integrity during development is poorly understood. The conserved small GTPase Rap1 is critical for establishing cell polarity and regulating cadherin-catenin cell junctions. Here, we identified a new role for Rap1 in maintaining epithelial integrity and tissue shape during Drosophila oogenesis. Loss of Rap1 activity disrupted the follicle cell epithelium and the shape of egg chambers during a period of major growth. Rap1 was required for proper E-Cadherin localization in the anterior epithelium and for epithelial cell survival. Both Myo-II and the adherens junction-cytoskeletal linker protein α-Catenin were required for normal egg chamber shape but did not strongly affect cell viability. Blocking the apoptotic cascade failed to rescue the cell shape defects caused by Rap1 inhibition. One consequence of increased cell death caused by Rap1 inhibition was the loss of polar cells and other follicle cells, which later in development led to fewer cells forming a migrating border cell cluster. Our results thus indicate dual roles for Rap1 in maintaining epithelia and cell survival in a growing tissue during development.

Expect the unexpected: conventional and unconventional roles for cadherins in collective cell migration.

Migrating cell collectives navigate complex tissue environments both during normal development and in pathological contexts such as tumor invasion and metastasis. To do this, cells in collectives must stay together but also communicate information across the group. The cadherin superfamily of proteins mediates junctional adhesions between cells, but also serve many essential functions in collective cell migration. Besides keeping migrating cell collectives cohesive, cadherins help follower cells maintain their attachment to leader cells, transfer information about front-rear polarity among the cohort, sense and respond to changes in the tissue environment, and promote intracellular signaling, in addition to other cellular behaviors. In this review, we highlight recent studies that reveal diverse but critical roles for both classical and atypical cadherins in collective cell migration, specifically focusing on four in vivo model systems in development: the Drosophila border cells, zebrafish mesendodermal cells, Drosophila follicle rotation, and Xenopus neural crest cells.

Occupancy maps of 208 chromatin-associated proteins in one human cell type.

Transcription factors are DNA-binding proteins that have key roles in gene regulation1,2. Genome-wide occupancy maps of transcriptional regulators are important for understanding gene regulation and its effects on diverse biological processes3-6. However, only a minority of the more than 1,600 transcription factors encoded in the human genome has been assayed. Here we present, as part of the ENCODE (Encyclopedia of DNA Elements) project, data and analyses from chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiments using the human HepG2 cell line for 208 chromatin-associated proteins (CAPs). These comprise 171 transcription factors and 37 transcriptional cofactors and chromatin regulator proteins, and represent nearly one-quarter of CAPs expressed in HepG2 cells. The binding profiles of these CAPs form major groups associated predominantly with promoters or enhancers, or with both. We confirm and expand the current catalogue of DNA sequence motifs for transcription factors, and describe motifs that correspond to other transcription factors that are co-enriched with the primary ChIP target. For example, FOX family motifs are enriched in ChIP-seq peaks of 37 other CAPs. We show that motif content and occupancy patterns can distinguish between promoters and enhancers. This catalogue reveals high-occupancy target regions at which many CAPs associate, although each contains motifs for only a minority of the numerous associated transcription factors. These analyses provide a more complete overview of the gene regulatory networks that define this cell type, and demonstrate the usefulness of the large-scale production efforts of the ENCODE Consortium.

Protein phosphatase 1 activity controls a balance between collective and single cell modes of migration.

Collective cell migration is central to many developmental and pathological processes. However, the mechanisms that keep cell collectives together and coordinate movement of multiple cells are poorly understood. Using the Drosophila border cell migration model, we find that Protein phosphatase 1 (Pp1) activity controls collective cell cohesion and migration. Inhibition of Pp1 causes border cells to round up, dissociate, and move as single cells with altered motility. We present evidence that Pp1 promotes proper levels of cadherin-catenin complex proteins at cell-cell junctions within the cluster to keep border cells together. Pp1 further restricts actomyosin contractility to the cluster periphery rather than at individual internal border cell contacts. We show that the myosin phosphatase Pp1 complex, which inhibits non-muscle myosin-II (Myo-II) activity, coordinates border cell shape and cluster cohesion. Given the high conservation of Pp1 complexes, this study identifies Pp1 as a major regulator of collective versus single cell migration.

[How many patient transfer rooms are necessary for my OR suite? : Effect of the number of OR transfer rooms on waiting times and patient throughput in the OR - analysis by simulation]. / Wie viele Patientenschleusen braucht mein OP-Bereich? : Einfluss der Zahl von OP-Schleusen auf Wartezeiten und Patientendurchsatz im OP - Analyse mithilfe einer Simulation.

INTRODUCTION: In most hospitals the operating rooms (OR) are separated from the rest of the hospital by transfer rooms where patients have to pass through for reasons of hygiene. In the OR transfer room patients are placed on the OR table before surgery and returned to the hospital bed after surgery. It could happen that the number of patients who need to pass through a transfer room at a certain point in time exceed the number of available transfer rooms. As a result the transfer rooms become a bottleneck where patients have to wait and which, in turn, may lead to delays in the OR suite. In this study the ability of a discrete event simulation to analyze the effect of the duration of surgery and the number of ORs on the number of OR transfer rooms needed was investigated. METHODS: This study was based on a discrete event simulation model developed with the simulation software AnyLogic®. The model studied the effects of the number of OR transfer rooms on the processes in an OR suite of a community hospital by varying the number of ORs from one to eight and using different surgical portfolios. Probability distributions for the process duration of induction, surgery and recovery and transfer room processes were calculated on the basis of real data from the community hospital studied. Furthermore, using a generic simulation model the effect of the average duration of surgery on the number of OR transfer rooms needed was examined. RESULTS: The discrete event simulation model enabled the analysis of both quantitative as well as qualitative changes in the OR process and setting. Key performance indicators of the simulation model were patient throughput per day, the probability of waiting and duration of waiting time in front of OR transfer rooms. In the case of a community hospital with 1 transfer room the average proportion of patients waiting before entering the OR was 17.9 % ± 9.7 % with 3 ORs, 37.6 % ± 9.7 % with 5 ORs and 62.9 % ± 9.1 % with 8 ORs. The average waiting time of patients in the setting with 3 ORs was 3.1 ± 2.7 min, with 5 ORs 5.0 ± 5.8 min and with 8 ORs 11.5 ± 12.5 min. Based on this study the community hospital needs a second transfer room starting from 4 ORs so that there is no bottleneck for the subsequent OR processes. The average patient throughput in a setting with 4 ORs increased significantly by 0.3 patients per day when a second transfer room is available. The generic model showed a strong effect of the average duration of surgery on the number of transfer rooms needed. CONCLUSION: There was no linear correlation between the number of transfer rooms and the number of ORs. The shorter the average duration of surgery, the earlier an additional transfer room is required. Thus, hospitals with shorter duration of surgery and fewer ORs may need the same or more transfer rooms than a hospital with longer duration of surgery and more ORs. However, with respect to an economic analysis, the costs and benefits of installing additional OR transfer rooms need to be calculated using the profit margins of the specific hospital.

The effect of optimal medical therapy on 1-year mortality after acute myocardial infarction.

OBJECTIVES: Five drug classes have been shown to improve the prognosis of acute myocardial infarction in clinical trials: aspirin, beta-blockers, statins, renin angiotensin system (RAS) blockers and thienopyridines. We aimed to assess whether the benefits of combining these drugs (termed optimal medical therapy, OMT), will result in a reduction of mortality in clinical practice. DESIGN: Nationwide registry SETTING: Hospitals with a cardiology unit or internal medicine department. PATIENTS: 5353 patients with acute myocardial infarction. At hospital discharge 89% received aspirin, 90% beta-blockers, 84% statins, 81% RAS blockers, 70% a thienopyridine and 46.2% OMT. INTERVENTIONS: Pharmacotherapy MAIN OUTCOME MEASURES: OR with 95% CI for mortality from myocardial infarction were calculated and adjusted for patient risk at baseline. RESULTS: Total mortality was reduced by 74% in patients receiving OMT (adj OR 0.26; 95% CI 0.18 to 0.38) versus patients receiving one or no drug. This was consistent in subgroups defined by STEMI/NSTEMI, diabetes and gender. Mortality was also reduced in patients receiving 2-4 drugs (adj OR 0.49; 95% CI 0.35 to 0.68), diabetic patients being the only subgroup with no significant effect. Analyses on the relative importance of either component revealed that withdrawal of beta-blockers (adj OR 0.63; 95% CI 0.34 to 1.16) and/or a combination of aspirin/clopidogrel (adj OR 0.59; 95% CI 0.20 to 1.17) abolished the risk reduction conferred by OMT. CONCLUSIONS: OMT over 1 year was associated with a significantly lower mortality of patients with acute myocardial infarction in clinical practice. However OMT is provided to less than half of eligible patients leaving room for substantial improvement.

[Reduction of in-hospital mortality and improved secondary prevention after acute myocardial infarction. First results from the registry of secondary prevention after acute myocardial infarction (SAMI)]. / Rückgang der Krankenhaus-Sterblichkeit und verbesserte Sekundärprävention nach akutem Myokardinfarkt. Erste Ergebnisse des SAMI-Registers.

BACKGROUND AND OBJECTIVE: In hospital mortality of acute myocardial infarction (AMI) has been reduced due to the availability of better therapeutic strategies. But there is still a gap between mortality rates in randomised trials and daily clinical practice. Thus, it was aim of the present registry to document the course and outcome of patients with AMI and to improve patient care by implementing recent guidelines. PATIENTS AND METHODS: In a nationwide registry study in hospitals in Germany with a cardiology unit or an internal medicine department data on consecutive patients were recorded for six to twelve months at admission, discharge and during a follow-up of one year. RESULTS: From 02/2003 until 10/2004 a total of 5,353 patients with acute myocardial infarction (65.7 % male, mean age of 67.6 +/- 17.7 years; 55.1 % of them with ST elevation myocardial infarction (STEMI) were included in the registry. Of the patients with STEMI, 76.6 % underwent acute intervention, 37.1 % had thrombolysis, 69.7 % percutaneous transluminal coronary angioplasty (PTCA). 40.0 % of those with non-Stemi (NSTEMI) had an acute intervention, 6.6 % thrombolysis, 73.5 % PTCA. Recommended secondary prevention consisted of ASS (93.2 %), beta-blockers (93.0 %), CSE-inhibitors (83.5 %), ACE-inhibitors (80.9 %) and clopidogrel (74.0 %). In-hospital mortality was 10.5 % (STEMI) and 7.4 % (NSTEMI). CONCLUSION: The 9 % mortality among patients with acute myocardial infarction treated in the hospitals participating in the SAMI registry is low compared to that in similar collectives. The high number of patients who had thrombofibrinolysis and coronary interventions as well as the early initiation of drug therapy contributed to these results. Medical treatment in the prehospital phase of these patients remains still insufficient and to a substantial extent contributes to the mortality of acute myocardial infarction.

Haplotype variation and linkage disequilibrium in 313 human genes.

Variation within genes has important implications for all biological traits. We identified 3899 single nucleotide polymorphisms (SNPs) that were present within 313 genes from 82 unrelated individuals of diverse ancestry, and we organized the SNPs into 4304 different haplotypes. Each gene had several variable SNPs and haplotypes that were present in all populations, as well as a number that were population-specific. Pairs of SNPs exhibited variability in the degree of linkage disequilibrium that was a function of their location within a gene, distance from each other, population distribution, and population frequency. Haplotypes generally had more information content (heterozygosity) than did individual SNPs. Our analysis of the pattern of variation strongly supports the recent expansion of the human population.

Role for GDNF in biochemical and behavioral adaptations to drugs of abuse.

The present study examined a role for GDNF in adaptations to drugs of abuse. Infusion of GDNF into the ventral tegmental area (VTA), a dopaminergic brain region important for addiction, blocks certain biochemical adaptations to chronic cocaine or morphine as well as the rewarding effects of cocaine. Conversely, responses to cocaine are enhanced in rats by intra-VTA infusion of an anti-GDNF antibody and in mice heterozygous for a null mutation in the GDNF gene. Chronic morphine or cocaine exposure decreases levels of phosphoRet, the protein kinase that mediates GDNF signaling, in the VTA. Together, these results suggest a feedback loop, whereby drugs of abuse decrease signaling through endogenous GDNF pathways in the VTA, which then increases the behavioral sensitivity to subsequent drug exposure.

Regulation of GluR2 promoter activity by neurotrophic factors via a neuron-restrictive silencer element.

The AMPA glutamate receptor subunit GluR2, which plays a critical role in regulation of AMPA channel function, shows altered levels of expression in vivo after several chronic perturbations. To evaluate the possibility that transcriptional mechanisms are involved, we studied a 1254-nucleotide fragment of the 5'-promoter region of the mouse GluR2 gene in neural-derived cell lines. We focused on regulation of GluR2 promoter activity by two neurotrophic factors, which are known to be altered in vivo in some of the same systems that show GluR2 regulation. Glial-cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) both induced GluR2 promoter activity. This was associated with increased expression of endogenous GluR2 immunoreactivity in the cells as measured by Western blotting. The effect of GDNF and BDNF appeared to be mediated via a NRSE (neuron-restrictive silencer element) present within the GluR2 promoter. The response to these neurotrophic factors was lost upon mutating or deleting this site, but not several other putative response elements present within the promoter. Moreover, overexpression of REST (restrictive element silencer transcription factor; also referred to as NRSF or neuron restrictive silencer factor), which is known to act on NRSEs in other genes to repress gene expression, blocked the ability of GDNF to induce GluR2 promoter activity. However, GDNF did not alter endogenous levels of REST in the cells. Together, these findings suggest that GluR2 expression can be regulated by neurotrophic factors via an apparently novel mechanism involving the NRSE present within the GluR2 gene promoter.

Enhancement of locomotor activity and conditioned reward to cocaine by brain-derived neurotrophic factor.

The mesolimbic dopamine (DA) system has been implicated in drug reward, locomotor sensitization, and responding for reward-related stimuli [termed conditioned reinforcers (CR)]. Here, we investigated the effect of brain-derived neurotrophic factor (BDNF), which enhances the survival and function of dopaminergic neurons, on stimulant-induced locomotor sensitization and responding for CR. In experiment 1, BDNF was infused into the nucleus accumbens (NAc) or ventral tegmental area over 2 weeks via chronically implanted minipumps (1-2.5 microgram/d), and the psychomotor stimulant effects of cocaine (5-15 mg/kg, i.p.) were studied. We found that BDNF enhanced the initial stimulant effects of cocaine and seemed to facilitate the development of sensitization to repeated cocaine doses. In experiment 2, we studied the effects of intra-NAc BDNF infusions on responding for CR. BDNF-treated rats showed twice as many CR responses compared with controls when saline was first administered. BDNF enhanced responding on the CR lever more than four times that seen in control animals after a cocaine injection (10 mg/kg, i.p.). The enhanced response to cocaine in BDNF-treated animals persisted for more than a month after the BDNF infusions had stopped, indicating long-lasting changes in the mesolimbic DA system caused by BDNF administration. In experiment 3, we examined locomotor sensitization to cocaine in heterozygous BDNF knock-out mice and found that the development of sensitization was delayed compared with wild-type littermates. These results demonstrate the profound effects of BDNF on the enhancement of both cocaine-induced locomotion and facilitation of CR and suggest a possible role for BDNF in long-term adaptations of the brain to cocaine.

Regulation of tyrosine hydroxylase promoter activity by chronic morphine in TH9.0-LacZ transgenic mice.

Levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, are known to be upregulated in specific brain regions by chronic administration of drugs of abuse. Chronic morphine administration increases TH levels in the locus coeruleus and ventral tegmental area, whereas chronic cocaine administration increases TH levels in the ventral tegmental area only. While such upregulation of TH has been related to behavioral effects of the drugs, the mechanism underlying these adaptations has remained controversial. To study the possibility that upregulation of TH occurs at the transcriptional level, we investigated the effect of chronic morphine or cocaine treatment on the activity of the TH gene promoter (9.0 kb), coupled to the LacZ reporter gene, in transgenic mice. These TH9.0-LacZ mice have been shown to exhibit correct tissue-specific expression and regulation of the reporter gene. We show here that chronic (but not acute) exposure of the TH9.0-LacZ mice to morphine increases the expression of beta-galactosidase (which is encoded by the LacZ gene) in the locus coeruleus by twofold compared with sham-treated mice. In contrast, beta-galactosidase expression in the ventral tegmental area was decreased 20-25% by chronic morphine and unaffected by chronic cocaine administration. Similar results were obtained after analysis of TH mRNA levels in these brain regions by in situ hybridization. These results suggest that chronic morphine upregulates TH expression via transcriptional mechanisms in the locus coeruleus but by post-transcriptional mechanisms in the ventral tegmental area.

Expression of messenger RNAs encoding ionotropic glutamate receptors in rat brain: regulation by haloperidol.

In situ hybridization was used to study the regional distribution of messenger RNAs encoding ionotropic glutamate receptor subtypes in the rat brain's dopaminergic cell body regions and their forebrain projection areas. Short oligonucleotide probes specific for the messenger RNAs encoding the flip or flop splice forms of the GluR1 and GluR2 AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptor subunits, or for the messenger RNAs encoding the N-methyl-D-aspartate R1 subunit, were used. Significant differences were seen in the relative messenger RNA levels, and the distribution of the flip and flop splice forms, of GluR1 and GluR2. In the dopaminergic cell groups of the substantia nigra pars compacta and the ventral tegmental area, the flip form of both GluR1 and GluR2 dominated over the flop form. Similarly, in the core division of the nucleus accumbens, GluR1 and GluR2 flip forms dominated over the flop forms. In contrast, in the accumbens shell, the GluR1 and GluR2 flop forms dominated over the flip forms. As a comparison to the AMPA receptor subunits, N-methyl-D-aspartate R1 messenger RNA was relatively evenly distributed in all the regions analysed. The results demonstrate a heterogeneous distribution of the flip and flop splice forms of GluR1 and GluR2 in the brain's dopaminergic pathways, which could contribute to physiological differences in regulation of the pathways by glutamatergic neurotransmission. We also studied regulation of glutamate receptor subunit expression in these regions by antipsychotic drugs, based on previous reports of altered levels of subunit immunoreactivity after drug treatment. Chronic administration of the typical antipsychotic drug, haloperidol, caused a small but significant induction of GluR2 flip messenger RNA in the dorsolateral caudate putamen. This effect was not seen after chronic administration of the atypical antipsychotic drug, clozapine. Significant drug regulation of the other glutamate receptor subunits studied was not observed.

Effect of composition of experimental solutions on neuronal survival during rat brain slicing.

Brain slices are widely used for experimentation; however, preparing brain slices results in significant injury as a result of a combination of ischemia prior to slicing and trauma during slicing, both of which are inevitable using this technique. The degree of injury is greater when using the recently developed approach for preparing thin slices for patch-clamp recording (9), presumably due to the greater degree of trauma. In cultured neurons, the events leading to death after exposure to combined anoxia and hypoglycemia (4, 11, 12, 15), or resulting from dendrotomy (21), are thought to be initiated by sodium and calcium influx. We have examined whether manipulations designed to block sodium and calcium influx are neuroprotective during preparation of thin (100 microM) brain slices, as a model of neuronal injury, as well as to help improve slice viability for electrophysiologic experimentation. Slices of the rat medulla were prepared using solutions with: (1) high osmolarity; (2) low calcium plus kynurenic acid; or (3) both. Slicing in Ringer resulted in immediate and marked neuronal swelling. After 4 h of incubation, there was nearly complete loss of neurons throughout the medulla. Preparation of slices using high osmolarity resulted in a marked decrease in the number of swollen neurons after slicing, but many neurons subsequently died over the next 2-3 h. Preparation of slices in zero calcium and kynurenic acid did not prevent swelling, but did result in a small increase in survival of neurons after 4 h. Preparation of slices in Ringer solution with a combination of high osmolarity, zero calcium, and kynurenic acid decreased both swelling and subsequent death, with survival of nearly as many neurons at 4 h as seen in brains perfused in situ with formalin. Similar results were obtained from the hippocampus and cerebral cortex. We hypothesize that the use of these solutions decreases neuronal damage by decreasing cytotoxic edema and calcium influx, suggesting that in this complex model of injury with a combination of trauma and ischemia, similar pathophysiologic mechanisms exist as during anoxic, hypoglycemic, and other forms of injury in cultured neurons.

Failure of repeated blood patch in a parturient with spinal headache.

An obstetric patient, who received and accidental dural puncture, developed symptoms that persisted until the administration of a third epidural blood patch. Her management is presented. The possible causes of failure of this technique are discussed, with specific reference to the obstetric patient. The role of cerebrospinal fluid in the epidural space at the time of patching is considered.

Hydralazine does not restore uterine blood flow during cocaine-induced hypertension in the pregnant ewe.

Cocaine abuse is widespread, and its use by the parturient has potential significant adverse effects in both the mother and the newborn. This study was undertaken in gravid ewes to determine the effects of treatment of cocaine-induced hypertension with hydralazine (Apresoline) on the maternal and fetal cardiovascular systems, catecholamine response, blood gas and acid-base status, and uterine blood flow (UBF). Twenty-one experiments were performed in 15 chronically instrumented ewes near term gestation. After a 30-min control period, cocaine was given intravenously to all ewes for 55 min to induce and maintain increased maternal mean arterial pressure (MMAP) and reduced UBF. The sheep were randomly assigned to receive either cocaine alone (n = 11, control group) or hydralazine (n = 10, treatment group), starting 15 min after the cocaine administration. Both drugs were discontinued 55 min after the start of the cocaine administration, followed by a 35-min recovery period. In the control group, cocaine administration resulted in a 31 +/- 13% (SD) increase in MMAP (P less than 0.05) and a 26 +/- 21% reduction in UBF (P less than 0.05). In the treatment group, the initial cocaine administration resulted in a similar increase in MMAP and decrease in UBF. Hydralazine therapy restored MMAP toward baseline after 20 min of administration, but UBF remained reduced (37 +/- 17%) throughout therapy (P less than 0.05) and recovery (18 +/- 13%) (P less than 0.05). The maternal heart rate increased maximally by 121 +/- 33% (P less than 0.05) after the administration of hydralazine, compared with a 14 +/- 21% increase (P less than 0.05) in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)