The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas over-expression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.
Objective This case report is designed to illustrate an uncommon presentation of osteoradionecrosis (ORN) of the temporal bone and a treatment method for bloody otorrhea from a pseudoaneurysm of the internal carotid artery (ICA).
Design This is a single patient case report
Setting University of Missouri-Columbia Hospital and Clinics.
Participants The report describes a patient with a history of hypopharyngeal squamous cell carcinoma (SCCA) who was previously treated with chemoradiation therapy and salvage bilateral neck dissections and then presented in a delayed fashion with profuse, episodic bloody otorrhea. Computed tomography (CT) was consistent with ORN of the temporal bone. The patient underwent emergent cerebral angiography. A pseudoaneurysm of the cervicopetrous ICA was confirmed to be the source of the patient's bloody otorrhea. The lesion was treated by endovascular sacrifice of the ICA using the two-catheter coiling technique.
Results The patient had no neurologic sequelae or further bleeding after treatment.
Conclusions Bloody otorrhea is an uncommon presentation of ORN. Sacrifice of the internal carotid can be considered as a treatment when the source is pseudoaneurysmal.
osteoradionecrosis; temporal bone; bloody otorrhea; endovascular coiling; radiation
Multiple observations suggest that certain parasitic infections can be oncogenic. Among these, neurocysticercosis is associated with increased risk for gliomas and hematologic malignancies. We report the case of a 71-year-old woman with colocalization of a metazoan parasite, possibly cysticercosis, and a WHO grade IV neuroepithelial tumor with exclusively neuronal differentiation by immunohistochemical stains (immunopositive for synaptophysin, neurofilament protein, and Neu-N and not for GFAP, vimentin, or S100). The colocalization and temporal relationship of these two entities suggest a causal relationship.
Dual energy CT imaging is expected to play a major role in the diagnostic arena as it provides material decomposition on an elemental basis. The purpose of this work is to investigate the use of dual energy micro-CT for the estimation of vascular, tissue, and air fractions in rodent lungs using a post-reconstruction three-material decomposition method. We have tested our method using both simulations and experimental work. Using simulations, we have estimated the accuracy limits of the decomposition for realistic micro-CT noise levels. Next, we performed experiments involving ex vivo lung imaging in which intact lungs were carefully removed from the thorax, were injected with an iodine-based contrast agent and inflated with air at different volume levels. Finally, we performed in vivo imaging studies in (n=5) C57BL/6 mice using fast prospective respiratory gating in end-inspiration and end-expiration for three different levels of positive end-expiratory pressure (PEEP). Prior to imaging, mice were injected with a liposomal blood pool contrast agent. The mean accuracy values were for Air (95.5%), Blood (96%), and Tissue (92.4%). The absolute accuracy in determining all fraction materials was 94.6%. The minimum difference that we could detect in material fractions was 15%. As expected, an increase in PEEP levels for the living mouse resulted in statistically significant increases in air fractions at end-expiration, but no significant changes in end-inspiration. Our method has applicability in preclinical pulmonary studies where various physiological changes can occur as a result of genetic changes, lung disease, or drug effects.
Micro-CT; dual energy; small animal imaging; lung
We hypothesised that patients with advanced disease or a cancer type that has a poor prognosis may be more likely to report anxiety and depressive symptoms after diagnosis; younger age and female gender may moderate these effects.
Patients (n=3850) were consecutively assessed with PSSCAN, a standardised, validated tool, at two large cancer centres between 2004 and 2009.
Female patients reported more anxiety and depressive symptoms (P=0.003 to P<0.001) compared with men and a healthy comparison group. Older age was associated with fewer anxiety (P=0.033 to P<0.001) and fewer depressive symptoms (P<0.001), but this was not true for lung cancer. Presence of metastases was associated with more anxiety symptoms in patients with gastrointestinal (P=0.044; R2Δ=0.001), lung (P=0.011; R2Δ=0.016), and prostate (P=0.032; R2Δ=0.008) cancer, but this was not true for breast cancer. Furthermore, early disease stage was associated with fewer depressive symptoms among older prostate cancer patients (P=0.021; R2Δ=0.008). Men with early lung cancer reported fewer anxiety (P=0.020; R2Δ=0.013) and depressive (P=0.017; R2Δ=0.016) symptoms than men with advanced disease or women.
As hypothesised, disease stage was directly associated with emotional distress, except for patients with breast cancer. Furthermore, age and gender moderated some of these effects.
anxiety; depression; disease stage; cancer type; gender; age
A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.
Placement of ventricular reservoirs is a common practice to treat various tumors of the central nervous system (CNS). Ventricular catheter-reservoir-associated edema has been noted in the literature, but a thorough review of this literature identified no articles that examine this particular complication in neurooncology patients, specifically. We report two cases of ventricular catheter-reservoir-associated edema in patients receiving treatment for CNS metastasis.
The CagA protein of Helicobacter pylori interacts with numerous cellular factors, and is associated with increased virulence and risk of gastric carcinoma. We present here the co-crystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.
Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends upon PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.
The complete hospital and community records of 77 women were randomly selected from 232 women who had relapsed breast cancer between 2000 and 2005. Scrutiny of all management activities revealed a total cost of £1 939 329 (mean per patient of £25 186, 95% CI £13 705–£33 821). The median survival from time of relapse was 40.07 months and the median total cost per patient was £31 402.62. Including the community cost of a relapse provides a more realistic figure for future cost-effectiveness analysis of adjuvant breast cancer therapies.
cost; breast cancer; relapse
We report an open-label, prospective, crossover study involving 184 post-menopausal women experiencing hot flushes on adjuvant tamoxifen (T). Six weeks after switching to an AI, the primary end point, hot flush score, improved by 47.3% (P<0.001) compared to those reported on T. The mean mood rating scale (MRS) score improved by 9.7% (P=0.01). The total mean combined FACT (b+es) score improved from 134.2 (95% CI ±2.96) to 143.5 (95% CI ±2.96 <0.001), and the endocrine subscale improved by 9.8% from 51.73 (95% CI ±1.38) to 57.34 (CI ±1.38, P<0.001). At 6 weeks, significantly more women chose to remain on an AI: 133 (72%), vs 40 (22%) (P<0.001) preferring T. At 3 months, 107 (58%) preferred to remain on an AI, 55(30%) on T, and 22 (12%) withdrew. The overall arthralgia rate at 3 months was 47% on AI and 30% on T (P=0.001). In all 182 (99%) women reported appreciating the opportunity to experience both drugs. These data suggest that if patients suffering significant adverse effects on T are given the opportunity to try an AI, this empowers them to prioritise relative side-effects, improving wellbeing in a significant proportion. These data also highlight the need for hospital follow-up in this intolerant cohort.
aromatase inhibitors; breast cancer; quality of life; tamoxifen intolerance
Microdeletions of 3q29 have previously been reported, but the postulated reciprocal microduplication has only recently been observed. Here, cases from four families, two ascertained in Toronto (Canada) and one each from Edinburgh (UK) and Leiden (Netherlands), carrying microduplications of 3q29 are presented. These families have been characterized by cytogenetic and molecular techniques, and all individuals have been further characterized with genome-wide, high density single nucleotide polymorphism (SNP) arrays run at a single centre (The Centre for Applied Genomics, Toronto). In addition to polymorphic copy-number variants (CNV), all carry duplications of 3q29 ranging in size from 1.9 to 2.4 Mb, encompassing multiple genes and defining a minimum region of overlap of about 1.6 Mb bounded by clusters of segmental duplications that is remarkably similar in location to previously reported 3q29 microdeletions. Consistent with other reports, the phenotype is variable, although developmental delay and significant ophthalmological findings were recurrent, suggesting that dosage sensitivity of genes located within 3q29 is important for eye and CNS development. We also consider CNVs found elsewhere in the genome for their contribution to the phenotype. We conclude by providing preliminary guidelines for management and anticipatory care of families with this microduplication, thereby establishing a standard for CNV reporting.
Fish passing through dams may be injured or killed despite advances in turbine design, project operations and other fish bypass systems. The six-degree-of-freedom (6DOF) Sensor Fish device is an autonomous sensor package that characterizes the physical conditions and physical stresses to which fish are exposed when they pass through complex hydraulic environments. It has been used to identify the locations and operations where conditions are severe enough to injure or kill fish. During the design process, a set of governing equations of motion for the Sensor Fish was derived and simulated to understand the design implications of instrument selection and placement within the body of the device. The Sensor Fish package includes three rotation sensors, three acceleration sensors, a pressure sensor, and a temperature sensor with a sampling frequency of 2,000 Hz. Its housing is constructed of clear polycarbonate plastic. It is 24.5 mm in diameter and 90 mm in length and weighs about 43 g, similar to the size and density of a yearling salmon smolt. The accuracy of the pressure sensor was determined to be within 0.2 psi. In laboratory acceptance tests, the relative errors of both the linear acceleration and angular velocity measurements were determined to be less than 5%. An exposure is defined as a significant event when the acceleration reaches predefined thresholds. Based on the different characteristic of acceleration and rotation velocities, the exposure event is categorized as either a collision between the Sensor Fish and a solid structure or shear caused by turbulence. Since its development in 2005, the 6DOF Sensor Fish has been deployed successfully at many major dams in the United States.
fish-friendly turbine; Sensor Fish; fish injury mechanism
Clinical practice guidelines are regarded as powerful tools to achieve effective health care. Although many countries have built up experience in the development, appraisal, and implementation of guidelines, until recently there has been no established forum for collaboration at an international level. As a result, in different countries seeking similar goals and using similar strategies, efforts have been unnecessarily duplicated and opportunities for harmonisation lost because of the lack of a supporting organisational framework. This triggered a proposal in 2001 for an international guidelines network built on existing partnerships. A baseline survey confirmed a strong demand for such an entity. A multinational group of guideline experts initiated the development of a non-profit organisation aimed at promotion of systematic guideline development and implementation. The Guidelines International Network (G-I-N) was founded in November 2002. One year later the Network released the International Guideline Library, a searchable database which now contains more than 2000 guideline resources including published guidelines, guidelines under development, "guidelines for guidelines", training materials, and patient information tools. By June 2004, 52 organisations from 27 countries had joined the network including institutions from Oceania, North America, and Europe, and WHO. This paper describes the process that led to the foundation of the G-I-N, its characteristics, prime activities, and ideas on future projects and collaboration.
Cell replacement therapy in Parkinson's disease depends on a reliable source of purified dopamine (DA) neurons (PDN) and the identification of factors relevant to their survival. Our goal was to genetically tag and purify by flow cytometry embryonic midbrain DA neurons from a transgenic mouse line carrying 11 kb of human tyrosine hydroxylase promoter driving expression of the enhanced green fluorescent protein (GFP) for studies in vivo and in vitro. A 99% purification of GFP+ cells was achieved. When transplanted into 6-hydroxydopamine-treated rat striatum, PDN survived, became well-integrated and produced recovery from amphetamine-induced motor behaviors. However, when grown in culture, PDN died within days of plating. No known growth factors prevented PDN death as did incubation with novel factors in glia/glial-conditioned media. We conclude that GFP-tagged DA neurons can be purified to homogeneity and can survive and function when grown with glial factors in vitro or after transplantation in vivo.
KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression.
Integrin β1 is both overexpressed and in an ‘active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin β1 deregulation we stably knocked-down integrin β1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin β1 is required for cell adhesion, cell spreading and invasion. However, integrin β1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin β1 knockdown tumours were significantly more encapsulated and less invasive.
integrin β1; vulval squamous cell carcinoma; invasion
A previous review of inbreeding in natural populations suggested that close inbreeding (inbreeding coefficient f = 0.25) is generally rare in wild birds and mammals. However, the review did not assess rates of moderate inbreeding (f = 0.125), which may make a rather larger contribution to overall inbreeding in a population. Furthermore, previous studies may have underestimated the prevalence of inbreeding in wild populations with incomplete pedigrees. By categorizing inbreeding events by the relationship of the parental pair, we suggest a simple method for estimating rates of close and moderate inbreeding from incomplete pedigree data. We applied this method to three wild populations of ruminants: red deer on Rum, Scotland, Soay sheep on Hirta, Scotland and reintroduced Arabian oryx on the Jiddat-al-Harasis, Oman. Although paternal half-sib pairs were the most common category of inbreeding in all three populations, there was considerable variation among populations in the frequencies of the various categories of inbreeding. This variation may be largely explained by differences in population size and dynamics, in maternal and paternal sibship size and in the overlap of reproductive lifespan of consecutive generations. Close and moderate inbreeding appear to be a routine part of breeding behaviour in these ruminant populations.
Aim—To investigate a possible outbreak of tuberculosis in a hostel for homeless men using IS6110 profiling, a polymerase chain reaction (PCR) based fingerprinting technique.
Methods—Eight cases of tuberculosis were diagnosed in residents of the hostel over a period of 28 months. To provide epidemiological data, a heminested inverse PCR (HIP) assay targeting the insertion sequence IS6110 together with its upstream flanking region was used to fingerprint the eight isolates of M tuberculosis under investigation.
Results—The HIP technique gave IS6110 profiles which showed that while three isolates were clearly distinct, the remaining five strains were indistinguishable, suggesting the latter were representatives of a single outbreak strain.
Conclusions—The HIP assay proved discriminatory and facilitated repeated testing for the direct comparison of strains as more patients presented over the protracted course of this outbreak.
Key Words: tuberculosis • epidemiology • molecular typing • IS6110
The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 μg of tobramycin ml−1or 1.0 μg of ciprofloxacin ml−1. After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 ± 0.18 for tobramycin and 1.42 ± 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 μm into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.
Evolutionary and conservation biologists have a long-standing interest in the consequences of inbreeding. It is generally recognized that inbred individuals may experience reduced fitness or inbreeding depression. By the same token, relatively outbred individuals can have greater than average fitness, i.e. heterosis. However, nearly all of the empirical evidence for inbreeding depression comes from laboratory or domestic species. Inbreeding depression and heterosis are difficult to detect in natural populations due to the difficulties in establishing pedigrees. An alternative method is to correlate heterozygosity, which is measured using genetic markers, with a trait related to fitness. The typically studied traits, such as juvenile survival and growth rates, either cover only early life or are weakly correlated with lifetime breeding success (LBS). In this paper we show that heterozygosity is positively associated with male and female adult LBS in a wild population of red deer (Cervus elaphus) on the Isle of Rum, Scotland. To the authors' knowledge, this is the first time that inbreeding depression and/or heterosis have been detected for a trait highly correlated with overall fitness in both sexes in a wild population.
The fitness consequences of inbreeding and outbreeding are poorly understood in natural populations. We explore two microsatellite-based variables, individual heterozygosity (likely to correlate with recent inbreeding) and a new individual-specific internal distance measure, mean d2 (focusing on events deeper in the pedigree), in relation to two measures of fitness expressed early in life, birth weight and neonatal survival, in 670 red deer calves (Cervus elaphus) born on the Isle of Rum between 1982 and 1996. For comparison, we also analyse inbreeding coefficients derived from pedigrees in which paternity was inferred by molecular methods. Only 14 out of 231 calves (6.1%) had non-zero inbreeding coefficients, and neither inbreeding coefficient nor individual heterozygosity was consistently related to birth weight or neonatal survival. However, mean d2 was consistently related to both fitness measures. Low mean d2 was associated with low birth weight, especially following cold Aprils, in which foetal growth is reduced. Low mean d2 was also associated with low neonatal survival, but this effect was probably mediated by birth weight because fitting birth weight to the neonatal survival model displaced mean d2 as an explanatory variable. We conclude that in the deer population fitness measures expressed early in life do not show evidence of inbreeding depression, but they do show evidence of heterosis, possibly as a result of population mixing. We also demonstrate the practical problems of estimating inbreeding via pedigrees compared with a direct marker-based estimate of individual heterozygosity. We suggest that, together, individual heterozygosity and mean d2, estimated using microsatellites, are useful tools for exploring inbreeding and outbreeding in natural population.