text:
The Activity System
Human activity is a complex evolutionary achievement. In a simplified fashion, its emergence may be depicted in three major steps. First, the animal form of activity (Figure 1) is an immediately collective and populational 'methodology of survival' of a species. Even this type of activity is not just passive adaptation - "species do not adapt to environments; they construct them." (Lewontin, 1982, p. 163)
Figure 1: General structure of the animal form of activity (Engeström, 1987, p.74)
In animal evolution, we witness ruptures in each of the three sides of the triangle depicted in Figure 1. The uppermost side of 'individual survival' is ruptured by the emerging utilization of tools, most clearly demonstrated by the anthropoid apes. The left hand side of 'social life' is ruptured by collective traditions, rituals and rules, originating at the crossing of adaptation and mating. The right hand side of 'collective survival' is ruptured by division of labor, influenced by the practices of breeding, upbringing and mating, and appearing first as the evolving division of labor between the sexes (Figure 2).
Figure 2: Structure of activity in transition from animal to human (Engeström, 1987, p. 76)
The breakthrough into human cultural evolution - into a specifically human form of activity - requires that what used to be separate ruptures or emerging mediators become unified determining factors. At the same time, what used to be ecological and natural becomes economic and historical. What used to be adaptive activity is transformed into consumption and subordinated to the three dominant aspects of human activity - production, distribution, and exchange (or communication). The model suggests the possibility of analyzing a multitude of relations within the triangular structure of activity. However, the essential task is always to grasp the systemic whole, not just separate connections (Figure 3).
Figure 3: The structure of human activity (Engeström, 1987, p. 78)
In the model, the subject refers to the individual or sub-group whose agency is chosen as the point of view in the analysis. The object refers to the 'raw material' or 'problem space' at which the activity is directed and which is molded and transformed into outcomes with the help of physical and symbolic, external and internal mediating instruments, including both tools and signs. The community comprises multiple individuals and/or sub-groups who share the same general object and who construct themselves as distinct from other communities. The division of labor refers to both the horizontal division of tasks between the members of the community and to the vertical division of power and status. Finally the rules refer to the explicit and implicit regulations, norms and conventions that constrain actions and interactions within the activity system.
The model may be concretized by means of an example. Consider the work activity of a physician working at a primary care clinic. The object of his work is the patients with their health problems and illnesses. The outcomes include intended recoveries and improvements in health, as well as unintended outcomes such as possible dissatisfaction, non-compliance and low continuity of care. The instruments include such powerful tools as X-rays, laboratory, and medical records - as well as partially internalized diagnostic and treatment-related concepts and methods. The community consists of the staff of the clinic, distinguished from other competing or collaborating clinics and hospitals. The division of labor determines the tasks and decision-making powers of the physician, the nurse, the nurse's aide, and other employee categories. Finally, the rules regulate the use of time, the measurement of outcomes, and the criteria for rewards.
Figure 4: The work activity of a primary care physician
The same primary health care activity will look quite different if we take the point of view of another subject in the community, for instance a nurse. Yet both subjects share the overall object - the patients and their health problems. An activity system is always heterogeneous and multi-voiced. Different subjects, due to their different histories and positions in the division of labor, construct the object and the other components of the activity in different, partially overlapping and partially conflicting ways.
There is constant construction and renegotiation within the activity system. Coordination between different versions of the object must be achieved to ensure continuous operation. Tasks are reassigned and redivided, rules are bent and reinterpreted.
There is also incessant movement between the nodes of the activity. What initially appears as object may soon be transformed into an outcome, then turned into an instrument, and perhaps later into a rule (Engeström, 1996). For instance, an unusual medical case first appears as a problem, is transformed into a successful diagnosis and treatment, the account of which is used instrumentally as a prototype or model for other similar cases, and is gradually sedimented and petrified into a rule requiring certain procedures in all cases that fit the category. On the other hand, rules may be questioned, reinterpreted and turned into new tools and objects.
Activity is a collective, systemic formation that has a complex mediational structure. An activity system produces actions and is realized by means of actions. However, activity is not reducible to actions. Actions are relatively short-lived and have a temporally clear-cut beginning and end. Activity systems evolve over lengthy periods of socio-historical time, often taking the form of institutions and organizations.
Collective activity is connected to object and motive, of which the individual subjects are often not consciously aware. Individual action is connected to a more or less conscious goal. Leont'ev (1978, p. 52) pointed out that the concept of object is already contained in the very concept of activity; there is no such thing as objectless activity. An object is both something given and something projected or anticipated. A thing or phenomenon becomes an object of activity as it meets a human need. This meeting is "an extraordinary act" (Leont'ev, 1978, p. 54). The subject constructs the object, "singles out those properties that prove to be essential for developing social practice" (Lektorsky, 1984, p. 137). In this constructed, need-related capacity, the object gains motivating force that gives shape and direction to activity. The object determines the horizon of possible goals and actions.
Below the collective activity and individual action, there is the level of automatic operations. Operations are dependent on the conditions in which the action is performed. Leont'ev (1978, p. 66) gives an example from learning to drive a car.
"Initially every operation, such as shifting gears, is formed as an action subordinated specifically to this goal and has its own conscious 'orientation basis'. Subsequently this action is included in another action, ... for example, changing the speed of the car. Now shifting gears becomes one of the methods for attaining the goal, the operation that effects the change in speed, and shifting gears now ceases to be accomplished as a specific goal-oriented process: Its goal is not isolated. For the consciousness of the driver, shifting gears in normal circumstances is as if it did not exist. He does something else: He moves the car from a place, climbs steep grades, drives the car fast, stops at a given place, etc. Actually this operation [of shifting gears] may, as is known, be removed entirely from the activity of the driver and be carried out automatically. Generally, the fate of the operation sooner or later becomes the function of the machine."
The three-level structure of activity proposed by Leont'ev is depicted in Figure 5.
Figure 5: The hierarchical structure of activity
An activity system does not exist in a vacuum. It interacts with a network of other activity systems. For example, it receives rules and instruments from certain activity systems (e.g., management), and produces outcomes for certain other activity systems (e.g., clients). Thus, influences from outside 'intrude' into the activity systems. However, such external forces are not a sufficient explanation for surprising events and changes in the activity. The outside influences are first appropriated by the activity system, turned and modified into internal factors. Actual causation occurs as the alien element becomes internal to the activity. This happens in the form of imbalance. The activity system is constantly working through contradictions within and between its elements. In this sense, an activity system is a virtual disturbance- and innovation-producing machine.
The primary contradiction of all activities in capitalist socio-economic formations is that between the exchange value and the use value within each element of the activity system. The work activity of a physician in primary medical care may again serve as an illustration. The primary contradiction in object of the doctor's work activity takes the form of patient as person to be helped and healed versus patient as source of revenue and profit (or on the flip side, as opportunity to profit by cutting costs). As Leont'ev (1981, p. 255) observed:
"The doctor who buys a practice in some little provincial place may be very seriously trying to reduce his fellow citizens' suffering from illness, and may see his calling in just that. He must, however, want the number of the sick to increase, because his life and practical opportunity to follow his calling depend on that."
The primary contradiction can be found by focusing on any of the elements of the doctor's work activity. For example, instruments of this work include a tremendous variety of medicaments and drugs. But they are not just useful for healing - they are above all commodities with prices, manufactured for a market, advertised and sold for profit. Every doctor faces this contradiction in his or her daily decision making, in one form or another.
As a new element enters into the activity system from outside, secondary contradictions appear between the elements. An example of a secondary contradiction in medical work would be that caused by the emergence of new kinds of objects, that is, patients and their medical problems. Conflicts emerge between the increasingly ambivalent and complex problems and symptoms of the patients and the traditional biomedical diagnostic instruments. Patients' problems increasingly often do not comply with the standards of classical diagnosis and classification of diseases. They require an integrated social, psychological and biomedical approach which may not yet exist.
A tertiary contradiction appears when a culturally more advanced object and motive is introduced into the activity. Such a tertiary contradiction arises when, say, practitioners of a medical clinic, using experiences from other clinics, design and adopt a new model for their work that corresponds to the ideals of a more holistic and integrated medicine. The new ideas may be formally implemented, but they are internally resisted by the vestiges of the old activity.
Quaternary contradictions are those that emerge between the changing central activity and its neighboring activities in their interaction. Suppose that a primary care doctor, working on a new holistic and integrated basis, refers the patient to a hospital operating strictly on a traditional biomedical model. Conflicts and misunderstandings easily emerge between these activity systems.
These four levels of contradictions may now be placed in appropriate locations in a schematic network of activities presented in Figure 6.
Figure 6: Four levels of contradictions in a network of human activity systems. (Click to see bigger fig.)
Level 1: Primary inner contradiction (double nature) within each constituent component of the central activity.
Level 2: Secondary contradictions between the constituents of the central activity.
Level 3: Tertiary contradiction between the object/motive of the dominant form of the central activity and the object/motive of a culturally more advanced form of the central activity.
Level 4: Quaternary contradictions between the central activity and its neighbor activities.
Inner contradictions of an activity system are "the principle of its self-movement and (...) the form in which the development is cast" (Il'enkov, 1977, p. 330). This means that new qualitative forms of activity emerge as solutions to the contradictions of the preceding form. This in turn takes place in the form of 'invisible breakthroughs', innovations from below.
"In reality it always happens that a phenomenon which later becomes universal originally emerges as an individual, particular, specific phenomenon, as an exception from the rule. It cannot actually emerge in any other way. Otherwise history would have a rather mysterious form. Thus, any new improvement of labour, every new mode of man's action in production, before becoming generally accepted and recognised, first emerges as a certain deviation from previously accepted and codified norms. Having emerged as an individual exception from the rule in the labour of one or several men, the new form is then taken over by others, becoming in time a new universal norm. If the new norm did not originally appear in this exact manner, it would never become a really universal form, but would exist merely in fantasy, in wishful thinking." (Il'enkov, 1982, p. 83-84)
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Concept Formation
in the Wild
Special issue of Mind, Culture, and Activity


text:
Marcham advice...(2 Posts)
ghumphreys Sun 07-Jul-13 19:10:34
used Fri 04-Oct-13 19:33:13
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I have spiceworks installed on two machines (same ver and port numbers). The first installation is quite outdated but contains information re many machine which are no longer on site hence is quite useful whilst the second installation contains inventory of othernew machines which are not on the first install.
Is there a way to sync data across both sites so that I can end up with one single collection of inventoried systems ? I do not want to lose data from either systems.
Kind Regards
This topic was created during version 5.0.
The latest version is 7.5.00101.
6 Replies
Apr 15, 2011 at 10:51 UTC
You can try this:
I would back up on whichever one is not your main installation and do a fresh install to complete the steps. After everything is configured, restore your database.
This one is not the best how-to on backing up and restoring but it should work for you. Just omit the parts about moving your installation.
Let me know if you need more help!
Apr 15, 2011 at 10:57 UTC
Thanks Alex but the problem I have is that both machines have a collection of data which is not one the other i.e. one has data regarding machines which can no longer come back onsite whilst the other has information contains inventory of another set of systems which again are new but never to return to the network.
It is not ideal scenario hence I am quite confused as to how to deal with this.
Apr 15, 2011 at 11:09 UTC
So you want to sync the data between the two and not have just one installation that contains all the data. I don't know if you can span SW installations just yet. Might want to ask someone from SpiceWorks how (if) you can make this possible.
Apr 15, 2011 at 11:16 UTC
Ideally I want to move all the information from installsystem2 to installsystem1 without losing inventory data from installsystem1 if that makes sense. I shall try to post this to spiceworks as per your suggestion. Thanks all the same for your inputs
Apr 15, 2011 at 11:29 UTC
The steps I posted should still work for you. What will happen after you set up the second SW installation to talk to the first SW installation and restore the database is the second installation will report all its database contents to the first installation, adding them to the database of the primary installation. Is this what you are wanting to do?
Anyone from SW (or anyone who's been here longer than me) care to weigh in?
Jun 8, 2011 at 9:18 UTC
I am still in need of an answer from the Spicework gurus re this issue. I do not want to lose data on either installations
This discussion has been inactive for over a year.
You may get a better answer to your question by starting a new discussion.


text:
Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy
The COP9/signalosome (CSN) is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells.
Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and β-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription.
Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex.
We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate that CSN2 loss results in a phenotype distinct from that of cells lacking CSN5 possibly as a consequence of altered CSN5 activity within a resultant CSN subcomplex. Our data present the first evidence for the sequential loss of F-box proteins upon CSN manipulation and are the first to identify a potential link between CSN function and autophagy.
The regulated expression and degradation of proteins are critical to all aspects of cell development and proliferation. The two main routes for eukaryotic intracellular protein clearance are the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. A key component involved in regulating degradation of proteins by the UPS is the COP9 signalosome (CSN). The CSN is an eight-subunit (CSN1-8) protein complex, highly conserved amongst eukaryotes
Degradation of cellular proteins by the 26S proteasome
Given the potential number and diversity of target proteins requiring CRL mediated ubiquitination for degradation, dynamic regulation of the CRL complex repertoire in a cell at any given time is essential. All cullins studied (Cul1-5) have been shown to be modified by neddylation
In this present study we have investigated the effects of knocking down CSN2 and CSN5 in the model K562 cell line, a model of human erythrocyte and megakaryocyte progenitors
CSN2 and CSN5 knockdown and resulting aberrant SCF activity
Plasmids encoding short hairpin RNA (shRNA) to either CSN2 or CSN5 were electroporated into K562 cells together with H-2Kk plasmid and transfected cells sorted by anti- H-2Kk magnetic beads. Three days post transfection, neither CSN2 nor CSN5 protein could be detected by western blot analysis in their respective knockdown protein extracts (Fig.
Figure 1
CSN2 and CSN5 knockdowns result in Cul-1 hyperneddylation, loss of Skp-2 and accumulation of p27
CSN2 and CSN5 knockdowns result in Cul-1 hyperneddylation, loss of Skp-2 and accumulation of p27. K562 cells were transiently transfected in the absence of plasmid DNA (mock), with pMACS Kk.II plasmid alone (HKK) or transiently co-transfected with HKK plasmid together with either vector control (shVC), CSN2 knockdown (shCSN2) or CSN5 knockdown (shCSN5) plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested for analysis. (A) CSN2 (left) and CSN5 (right) protein knockdown was determined by western blot in three independent transfectants. (B) mRNA levels in vector control and knockdown cells were determined by QRT-PCR (CSN2, left, P = 0.0012; CSN5, right, P = 0.000031). Data represents 3 independent sets of triplicate transfections. (C) CSN5 and CSN2 protein levels were determined by western blot in n = 3 CSN2 (top) and CSN5 (bottom) knockdowns, respectively. (D) CSN5 mRNA levels in n = 3 vector control and CSN2 knockdown cells were determined by QRT-PCR (P = 0.011). (E) The level of Cul1 neddylation (top panels, † indicates neddylated Cul-1), Skp2 protein (second panels) and p27 protein (third panels) was determined in n = 3 shCSN2 (left) and shCSN5 (right) samples by western blot. Even gel loading was determined by β-actin signal. Graphical data indicates the mean ± s.e.m. * indicates significance with a p value of less than 0.05.
Additional file 1
Vector control scramble sequence has no effect on protein levels, mRNA expression or cell growth, whilst CSN5 knockdown resulted in the sequential loss of F-box proteins. K562 cells were transiently co-transfected with HKK plasmid together with either empty vector or vector control scramble plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested day 9 post transfection. (A) The levels of CSN2, CSN5, Cul1, Skp2, p27, LC3-II and caspase-9 protein was determined by western blot. (B) CSN2 and CSN5 mRNA levels in vector control scramble cells relative to empty vector transfected cells were determined by QRT-PCR. Data is the mean of n = 3 transfections ± s.e.m. The dashed line indicates mRNA expression in empty vector transfected cells. (C) Cell counts were taken daily and the cumulative growth calculated. The cumulative growth of shVC scramble cells is shown relative to empty vector transfected cells. Data shown are the mean ± s.e.m. of n = 3. (D) shVC and shCSN5 cells were harvested day 4 post transfection and the level of Skp-2, Cdc4, β-TrCP and β-actin protein determined by western blot.
Click here for file
The instability of particular CSN subunits in the absence of another subunit has been reported previously. For example, CSN8 knockdown has been shown to result in the loss of CSN3, CSN5 and CSN7 protein
Consistent with previous studies of CSN deregulation
Loss of CSN2 and CSN5 results in the sequential loss of F-box proteins
Skp2 is one of many F-box proteins that can bind to the Cul1/Roc1/Skp1 complex, thereby altering the substrate specificity of the SCF (F-box proteins reviewed in
Figure 2
Loss of CSN2 and CSN5 results in sequential loss of F-box proteins
Loss of CSN2 and CSN5 results in sequential loss of F-box proteins. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) shVC and shCSN2 cells were harvested day 6 and 9 post transfection and the level of Skp2, Cdc4, β-TrCP and β-actin protein determined by western blot. (B) The level of Skp2, Cdc4 and β-TrCP proteins (normalised for loading using β-actin) in shCSN2 at each time point was normalised to expression in shVC cells and the data plotted as the mean ± s.e.m. (C) shVC, shCSN2 and shCSN5 cells were treated with DMSO (control) or the proteasome inhibitor MG132 (10 μM) for the final 18 hours of culturing and the level of Skp-2, Cdc4, β-TrCP and β-actin protein determined by western blot. (D, E) The level of Skp2, Cdc4 and β-TrCP mRNA in shCSN2 (D) and shCSN5 (E) cells was determined at each time point post transfection relative to expression in vector control scramble cells by QRT-PCR. Data shown is the mean ± s.e.m of n = 3 transfections. * indicates a significant difference to vector controls with a p value of less than 0.05.
In order to determine the mechanism of F-box protein loss in both CSN2 and CSN5 knockdown cells, we investigated the rescue of F-box proteins by the proteasome inhibitor MG132 (Fig.
Both CSN2 and CSN5 knockdowns result in reduced cell growth and cell death
Knockdown of either CSN2 or CSN5 dramatically diminished cell proliferation, followed by loss of cell viability (Fig.
Figure 3
Both CSN2 and CSN5 knockdowns result in reduced cell growth
Both CSN2 and CSN5 knockdowns result in reduced cell growth. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) Cell counts were taken daily and the cumulative growth calculated. The cumulative growth of shCSN2 and shCSN5 cells is shown relative to shVC. The insert graph has a different Y scale to highlight the differences between shCSN2 and shCSN5 cumulative growth profiles. Data shown are the mean ± s.e.m. of n = 3. * indicates a significant difference to shVC cell growth with a p value of less than 0.05. (B) Thymidine incorporation in shCSN2 cells relative to shVC cells was measured day 3, 5 and 7 post transfection. Data shown are the mean ± s.e.m. of n = 3. (C) Thymidine incorporation in shCSN5 cells relative to shVC cells was measured day 3 and 4 post transfection. Data shown are the mean ± s.e.m. of n = 3. * indicates significance with a p value of less than 0.05.
Knockdown of CSN5 but not CSN2 is associated with cell cycle arrest and defects in mitotic spindle formation
Cell cycle analyses revealed striking differences between the profiles of CSN2 and CSN5 knockdown cells. Despite leading to significantly reduced cell proliferation, loss of CSN2 did not affect the cell cycle distribution of K562 cells as compared to shVC K562 cells by day 6 post transfection (Fig.
Figure 4
Cell cycle arrest in CSN5 knockdowns is associated with mitotic defects
Cell cycle arrest in CSN5 knockdowns is associated with mitotic defects. K562 cells were transiently co-transfected with HKK plasmid together with shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) Representative images of shCSN2 (left, dark grey line) and shCSN5 (right, dark grey line) cell cycle profiles relative to shVC cells (light grey fill). Pie charts show data as the mean ± s.e.m. of n = 3. * indicates significance with a p value of less than 0.05. (B) Cytospins of shVC and shCSN5 cells were stained by Jenner-Giemsa day 4 post transfection. (C) Cytospins were immunostained for tubulin (FITC) and Hoescht (blue) for visualisation of DNA in shVC and shCSN5 cells day 4 post transfection. All images shown are representative of n = 3 transfections.
Knockdown of CSN5 but not CSN2 results in apoptotic cell death
In order to determine whether the subsequent loss of viability in CSN2 and CSN5 knockdown cells was due to apoptosis, cells were co-analysed for annexin V staining and propidium iodide uptake day 7 and day 6 post transfection, respectively (Fig.
Figure 5
CSN5, but not CSN2, knockdown results in apoptotic cell death
CSN5, but not CSN2, knockdown results in apoptotic cell death. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Binding of Annexin V and uptake of propidium iodide were analysed by flow cytometry. The lower left quadrant encompasses the viable population of cells, the lower right quadrant contains early apoptotic cells, the upper right quadrant contains late apoptotic cells and the upper left quadrant contains the necrotic cell population. Dot plots shown are representative of n = 3 transfections. The mean of three data sets was taken and the values shown in the corresponding quadrant ± s.e.m. * indicates significance with a p value of less than 0.05. (B) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of caspase-9 cleavage determined by western blot. (C) Propidium iodide uptake was determined day 6 post transfection. The histogram shown is representative of n = 3 shVC (light grey in fill), shCSN2 (dark grey line) and shCSN5 (black line). The significant shift in propidium iodide staining in shCSN2 cells is shown ± s.e.m. * indicates significance with a p value of less than 0.05.
CSN2 but not CSN5 knockdown is associated with autophagy
Whilst culturing the transfectants, marked differences in cell morphology were noted in the CSN2 knockdown cells. Jenner-Giemsa staining of cytospins identified large vacuoles in a significant proportion of CSN2 knockdown cells which were not present in either shVC or shCSN5 cells (Fig.
Figure 6
Cells lacking CSN2, but not CSN5, are associated with autophagy
Cells lacking CSN2, but not CSN5, are associated with autophagy. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Jenner-Giemsa staining of shVC and shCSN2 cytospins day 6 post transfection. (B) shVC and shCSN2 cells were stained with the autophagosome marker monodansylcadaverine day 6 post transfection. All images shown are representative of n = 3 transfections. (C) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of LC3-II protein determined by western blot. Even loading was determined by β-actin signal. (D) Cell counts were taken daily and the cumulative growth of shVC cells and shCSN2 cells +/- 3-MA calculated. The insert has had the untreated shVC data removed in order to demonstrate the similarity between the shVC +3-MA and shCSN2 cumulative growth profiles. Data shown is the mean of n = 3 ± s.e.m. (E) Jenner-Giemsa staining of shVC +/- 1 μM bafilomycin A1 cytospins day 7 post transfection. Images shown are representative of n = 3 transfections. (F) Cells were harvested day 7 post transfection and the level of LC3-II and β-actin protein determined by western blot.
Loss of CSN2 results in the formation of an alternative CSN5 containing complex
The above data indicate that knockdown of CSN2 or CSN5 in K562 cells results in common derangement of SCF activity as measured by accumulation of hyperneddylated Cul1, accumulation of p27 and the sequential loss of Skp-2, cdc4 and β-TrCP. However, the cellular responses to CSN2 or CSN5 knockdown were markedly different. Since it has previously been shown that depletion of individual subunits can destabilize the CSN holocomplex
Figure 7
Loss of CSN2 results in the formation of CSN5 containing CSN subcomplexes
Loss of CSN2 results in the formation of CSN5 containing CSN subcomplexes. K562 cells were transiently co-transfected with HKK plasmid together with either shVC or shCSN2 plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested day 3 post transfection. Both CSN2 (A) and CSN5 (B, short exposure; C, long exposure) distribution in shVC and shCSN2 cells was determined by 2-Dimensional Native-PAGE/SDS-PAGE and western blot analysis. All data shown is representative of n = 3 transfections.
Immunoblotting of extracts from shVC cells with either CSN2 or CSN5 antibody identified 2 major complexes, one of approximately 500 KDa, correlating in size with the CSN holocomplex, and the second of 750 KDa (Fig.
The achievement of almost complete CSN2 and CSN5 knockdown in this study has provided a powerful tool to study the function of these CSN subunits more closely. At the molecular level, CSN2 and CSN5 knockdowns resulted in aberrant SCF activity, with the accumulation of neddylated Cul-1, loss of the F-box protein Skp2 and an increase in the Skp2 target protein, p27. This complements another report in which CSN4 and CSN5 knockdown also resulted in increased neddylation of Cul-1 with a concomitant loss of Skp2 and increase in p27 protein in human epithelial cell lines rather than haemopoietic cells
It was also observed that CSN2 knockdown not only results in loss of CSN5 protein but also results in a significant reduction of CSN5 mRNA. Moreover, both CSN2 and CSN5 knockdown resulted in temporal alterations of F-box protein mRNA. Together with other recent reports
Here we show for the first time sequential loss of F-box proteins following knockdown of CSN subunits. Moreover, protein levels were at least partially restored in both knockdowns upon treatment with the proteasome inhibitor MG132. These observations are in accordance with the finding that F-box proteins are autocatalytically degraded in the presence of hyperneddylated Cul-1
The CSN5 knockdown cultures contained aberrantly large cells and were associated with G2/M arrest and apoptosis. This data complements previous studies demonstrating that CSN5 loss inhibits proliferation and induces apoptosis
K562 cells in which CSN2 had been knocked down did not display apoptosis markers as in CSN5 knockdown cells, but were instead associated with features of autophagy. Interestingly, autophagy inhibitors recapitulated the cell growth kinetics, vacuolar morphology and LC3-II accumulation of cells lacking CSN2, whilst treatment of CSN2 knockdown cells with one of these inhibitors (3-MA) had a comparatively mild effect on cell growth. These findings suggest that CSN2 knockdown K562 cells undergo autophagy inhibition resulting in non-apoptotic cell death. This is the first data to show an association between the CSN complex and autophagy.
The distinct phenotypes observed between CSN2 and CSN5 knockdowns may arise as a result of aberrant CSN5 activity within the observed CSN subcomplex in cells lacking CSN2. However, it is important to note that both the CSN subunits studied here have CSN independent functions
In conclusion, we have shown that loss of either CSN2 or CSN5 in human K562 cells results in significant loss of viability but by very different mechanisms, potentially attributable to the formation of a CSN5 containing subcomplex in the absence of CSN2. Furthermore, we have provided data to suggest a possible function of the CSN complex in the transcriptional regulation of both its own components and CRL subunits. Finally, we have demonstrated here for the first time the sequential loss of F-box proteins in the absence of the CSN complex and have provided the first evidence of a link between the CSN complex and autophagy.
Cell culture and treatments
K562 cells were cultured in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% v/v foetal bovine serum (Invitrogen, Gibco) and maintained at 37°C with 5% CO2. For proteasome inhibition, cells were treated with 10 μM MG132 for the final 18 hours of culturing. For autophagy inhibition, cells were treated in culture with either 10 mM 3-methyladenine from day 3-day 7 post transfection or 1 μM bafilomycin A1 for 48 hours day 5-day 7 post transfection.
shRNA constructs
The shRNA vector used was a modified pcDNA3.1 vector (pcDNA3.1-H1) developed by Heiko Lickert
CSN2 knockdown 5'AAGCGGCATTAAGCAGTTTCC3'
CSN5 knockdown – 5'AAGGGCTACAAACCTCCTGAT3'
shRNA scramble control – 5'AAGCGGGATTCAGTAGTTACG3'
Transfections and cell sorting
Transfection efficiencies in K562 cells vary between 20–50%. Therefore, to allow enrichment of transfected cells, 5 × 106 K562 cells were electroporated in Nucleofector kit V solution (Amaxa) using a Nucleofector I (Amaxa) and file T16, with 5 μg pMACS Kk.II and 10 μg of the relevant knockdown pcDNA3.1-H1 plasmid according to manufacturer guidelines. The pMACS Kk.II produces a truncated murine MHC class I cell surface protein, H-2Kk, which lacks the cytoplasmic domain and is transiently expressed on the cell surface of transfected cells between 6 and 48 hours post-transfection. Transfected cells were sorted 24 hours post transfection using anti-H-2Kk antibody conjugated to magnetic beads, MACS MS columns and a MACS magnet (Miltenyi Biotec) according to manufacturer instructions. Post sorting, cells were set at 3 × 105/ml daily and cells harvested for protein and mRNA analysis as indicated in results.
Thymidine incorporation assay
2 × 104 cells were pulsed with 2 μCi/ml 3H-thymidine (Amersham) for the final 18 hours of culture leading up to each time point. Samples were transferred to a filter mat (Wallac) using a Skatron cell harvester (Skatron Instruments) and read using a beta-plate scintillation counter (Skatron Instruments).
Immunofluorescence and Jenner-Giemsa staining
Cytospins were made with 5 × 104 cells in 80 μl, using a Shandon cytospin 3 (Shandon). For immunofluorescence staining, cytospins were fixed in 4% paraformaldehyde and stained using anti-β-tubulin antibody (Sigma, 1/500 dilution) followed by FITC labelled secondary antibody (Jackson Laboratories, 1/500 dilution). DNA was counterstained using Hoescht 33342 (Sigma, 1/1000 dilution). All reagents were diluted in PBS and slides mounted using Mowiol (6 g glycerol, 2.4 g Moviol-4-88 (Sigma), 12 ml 0.2 M Tris HCl pH8.5, anti-fade crystal (Sigma), 6 ml distilled water). Slides were viewed using an Axioskop2 microscope (Zeiss) and images captured with a Q-imaging 12-bit QICAM (Media Cybernetics) and Openlab software (Improvision).
For Jenner-Giemsa staining, cytospins were air-dried, methanol fixed and stained; First with Jenner staining solution (VWR, UK) diluted 1/3 in 1 mM sodium phosphate buffer pH5.6 (5 mins) and second with Giemsa stain (VWR, UK) diluted 1/20 in 1 mM sodium phosphate buffer pH5.6 (10 mins). Slides were dried and then mounted onto coverslips using DePex (VWR, UK). Slides were viewed with an Olympus BX40 microscope (Olympus) and images captured using an Olympus Chameleon digital SLR (Olympus).
Staining of autophagosomes
For visualisation of autophagic vacuoles, 5 × 104 cells were incubated with 0.05 mM monodansylcadaverine (MDC, Sigma) in 0.5 ml PBS for 10 minutes at 37°C. Cells were washed four times with PBS, cytospins made as above and cells viewed immediately using a Leica DMIRE2 system.
Fluorescence flow cytometry
For Annexin V labelling, 1 × 105 cells were stained using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer instructions, and staining analysed within 1 hour by flow cytometry. For cell cycle analysis 1 × 105 cells were resuspended in cell cycle buffer (10 μg/ml propidium iodide, 0.1 mM sodium chloride, 1% Triton X100) and samples analysed within 24 hours by flow cytometry. All staining was analysed using a FACS Calibur (Becton Dickinson) and the data evaluated using Cell Quest Pro software (Becton Dickinson).
Western blot analysis
Whole cell lysates were prepared using RIPA buffer (1% v/v NP40, 0.5% w/v sodium deoxycholate, 0.1% w/v 10% SDS, in distilled water) and protein quantified using the Dc protein assay according to manufacturer instructions (Bio-Rad). Forty micrograms of protein were boiled for 10 minutes in 1× SDS gel loading buffer (15.6 mM Tris HCl pH6.8, 6.25% v/v glycerol, 0.5% SDS, 1.25% v/v 2-mercaptoethanol, Bromophenol Blue, in distilled water). Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma). Proteins recognized by these antibodies were detected using ant-mouse (Sigma, 1/1000 dilution) or anti-rabbit (Pierce, 1/1000 dilution) HRP conjugated secondary antibody followed by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and autoradiography (Kodak X-Omat LS film, Sigma). Quantitative analysis of western blots was carried out using ImageJ software
2-Dimensional gel analysis
Native protein extracts were obtained from 2.5 × 105 cells by resuspending cells in 50 μl mild lysis buffer (25% 4× NativePAGE sample buffer (Invitrogen), 1% digitonin, 10% 10× protease inhibitor, in distilled water). Extracts were separated out in the first dimension using a NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to manufacturer instructions. The gel was then cut into individual lanes, proteins denatured by incubation in 1× SDS gel loading buffer and resolved in the second dimension by electrophoresis through 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane (Millipore) and immunoblotting performed as above.
Quantitative real-time PCR analysis (QRT-PCR)
RNA was extracted using the Qiagen RNeasy kit according to manufacturer instructions and cDNA generated using 1 μg RNA, random hexamers (Promega) and Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out using either TAQMAN or SYBR-Green based assays. For TAQMAN assays, QRT-PCR was carried out in duplicate 20 μl reactions containing 1× qPCR Mastermix Plus (Eurogentec), 20–40 ng cDNA, 18 pmoles each primer and 2.5 pmoles FAM/TAMRA dual labeled probes. For SYBR-Green assays, QRT-PCR was carried out in duplicate 25 μl reactions containing 1× Sensimix (Quantace), 20–40 ng cDNA, 9 pmoles each primer, 1× SYBR-Green solution (Quantace), 4 mM MgCl2 and 0.5 units UNG (Quantace). QRT-PCR was carried out on an ABI Prism 7000 sequence detector (Applied Biosystems). The following primers (Sigma Genosys) and FAM/TAMRA labeled probes (Eurogentec) were used:
5'-CATCATAATTCTTGAAGGCTTCAAAA-3' (reverse),
5'-CCCTCAAGTGCATTTTACCACCACATTCTCT-3' (probe);
5'-GGTCCTTCATCAGGAGGTTTGT-3' (reverse),
5'- TGGCGCCTTTAGGACATACCCAAAGG-3' (probe);
5'-CCATGTGCTGTACACGAAAAGG-3' (reverse);
5'-ACTCCAGCTCTGAAACATTTTTAGC-3' (reverse);
5'-TGTCCCATAATCTGATAGTGTTGTCA-3' (reverse)
5'-CATTCTTGGCAAATGCTTTCG-3' (reverse).
Preoptimised primers and probes to 18S ribosomal RNA were used as internal standards in TAQMAN QRT-PCR (Applied Biosystems). Cycle threshold (Ct) values were obtained graphically for test genes and 18S internal standards. ΔCt values were calculated by subtracting 18S Ct from test gene Ct, and average ΔCt values obtained from duplicates. Relative mRNA levels were determined by subtraction of mock transfection ΔCt values from shVC/shCSN2/shCSN5 ΔCt values to give a ΔΔCt value and conversion through 2-ΔΔCt.
Authors' contributions
CP conceived of and designed the study, carried out all laboratory experimentation except for that carried out by REH and FLK, and drafted the manuscript. REH harvested thymidine plates, generated the propidium iodide flow cytometry data and assisted in the analysis of all flow cytometry data. CMB and FLK conceived of and designed the study and drafted the manuscript. FLK designed the shRNA sequence and generated the plasmid for CSN2 knockdown. All authors read and approved the manuscript.
We would like to acknowledge Heiner Schrewe (MPI, Berlin), Debbie Cunningham (Univ of Birmingham, UK) and Simon Johnston (Univ of Birmingham, UK) for technical assistance. CP is funded by a grant from the BBSRC (UK) and REH and FLK are funded by a Leukaemia Research (UK) grant.
Characterization and purification of the mammalian COP9 complex, a conserved nuclear regulator initially identified as a repressor of photomorphogenesis in higher plants
The COP9/signalosome complex is conserved in fission yeast and has a role in S phase
The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis
Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans
The COP9 signalosome regulates cell proliferation of Dictyostelium discoideum
The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch
COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases
COP9 signalosome: a provider of DNA building blocks
The ubiquitin ligase activity in the DDB2 and CSA complexes is differentially regulated by the COP9 signalosome in response to DNA damage
Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates
Proteasomes: protein degradation machines of the cell
Proteolysis. The proteasome: a protein-degrading organelle?
The 26S proteasome: a molecular machine designed for controlled proteolysis
A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein
Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown
Function and regulation of cullin-RING ubiquitin ligases
SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box
F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex
F-box proteins: the key to protein degradation
Cullin-RING ubiquitin ligases: global regulation and activation cycles
Covalent modification of all members of human cullin family proteins by NEDD8
An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells
NEDD8 recruits E2-ubiquitin to SCF E3 ligase
Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity
Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome
Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1
Protection of cullin-RING E3 ligases by CSN-UBP12
Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels
CSN facilitates Cullin-RING ubiquitin ligase function by counteracting autocatalytic adapter instability
Identification and characterization of a novel zinc finger protein (HZF1) gene and its function in erythroid and megakaryocytic differentiation of K562 cells
A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis
The COP9 signalosome negatively regulates proteasome proteolytic function and is essential to transcription
The COP9 signalosome regulates Skp2 levels and proliferation of human cells
p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27
Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing
How to interpret LC3 immunoblotting
The COP9 signalosome (CSN): an evolutionary conserved proteolysis regulator in eukaryotic development
Cop9 signalosome subunit 8 (CSN8) is essential for Drosophila development
Downregulation of COP9 signalosome subunits differentially affects the CSN complex and target protein stability
The COP9 signalosome: at the interface between signal transduction and ubiquitin-dependent proteolysis
Retinoblastoma protein regulation by the COP9 signalosome
Depletion of Jab1 inhibits proliferation of pancreatic cancer cell lines
Targeted inactivation of the COP9 signalosome impairs multiple stages of T cell development
The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability
Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability
EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A
A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells
JAB1/CSN5 and the COP9 signalosome. A complex situation
A new group of conserved coactivators that increase the specificity of AP-1 transcription factors
Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor
The coregulator Alien
CSN5 binds to misfolded CFTR and promotes its degradation
The COP9 signalosome: more than a protease
The Jab1/COP9 signalosome subcomplex is a downstream mediator of Bcr-Abl kinase activity and facilitates cell-cycle progression
Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype


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Ectopic pregnancy support thread(909 Posts)
Hi everyone, this is a place for anyone who's had an ectopic pregnancy - whether you want to moan, cry, fret or just have a chat with other people who understand. I know there's lots of other good websites on ectopic pregnancies but after
hijackingchatting with sunshine on another thread, I thought I'd start one here.
Well I'll get the ball rolling with my story - after a MMC in November (found out at the 12 wk scan that the baby had died at 10 wks) I was just getting myself sorted, having counselling, when I had the ectopic. I know I am lucky in that I have a lovely DD. Having had a normal pregnancy, I knew from the beginning this one was not good and as soon as I got a BFP I was down at the hospital.
After 10 days of mental torture, waiting around, scans, rescans, blood tests, it got too late for the methotrexate (they finally saw the ectopic in a scan on a Friday early evening and the methotrexate wasn't in stock )
I finally had the surgery to remove one tube on the Tuesday. This was the end of February, at first I wasn't sure how I would cope, but I am just plodding on. I know I am lucky that it didn't rupture, but honestly I can't think of having an ectopic as being lucky. Especially after a MMC .
Anyway, come join me if you want to share your story, I have , and
Anybody out there listening i need 2 talk 2 some1 pls reply
Hi tearyeyes how are you feeling now? It's hard not to have support from your DH, is there anyone else you can talk to?
It's a bit quiet on here now, but I'm around, off load as and when you need to x
Hi tas thanks 4 responding im not good at all it seems like ppl whom i try 2 talk 2 dont understand me at all and im frustrated cos the dh does'nt seem 2 c how bad i feel he even abuse me emotionally where can i find counselling maybe dat wil help? Things are bad i cry a lot i try 2 hide my pain bt its too much and my operation scar its stil painful a lot is dat normal sorry im long
Im thankful i found this page talking 2 u may heal me
teary you need to go to your GP tomorrow, they will be able to check your scar and they should have an idea of where to go for counselling in your area. I went to one based at my hospital first, then I found a group of psychotherapists. Don't be afraid to try one out and not continue if you don't 'gel'. I tried a couple before I found one I liked. You should ask if they have experience in pg loss and also emotional abuse. Have you posted on the 'relationships' board about your DH? I don't have any experience with emotional abuse, but there are many people on there that can help. It sounds like you have too much to deal with right now, what can you do right now to take care of yourself and relax?
How long ago was your op? Open surgery takes about 6 weeks to heal. I found when I ovulated everything hurt more. Did you have a follow up appt at the hospital? Best to see your GP if you're worried.
Hi tas i'll go 2 gp and maybe he'll help me and refer me 2 counselling and emotionally im worse last week they tried 2 give us counselling after i tried commiting suicide things were better bt 2 days later it was back 2 square 1 the abuse started worse part he's cheating and shows me photos of her galfrind saying she's his frnds wife asking me if she's beautiful this is 2 much 4 me 1st was 2 ectopics nw this "crying"
Hi babybluebell,
I am sorry that you had to go through this pain. I understand because my situation is nearly the same as yours. I was 43 when I had my first pregnancy. I didn't realize I was pregnant at first. My period was 7 days late and when it started I had terrible cramps (worse than any cramp I ever experienced). It got worse by the day. I went to the A&E twice. The first was because I collapsed with the most excruciating abdominal pain. At my request, the first A&E did a pregnancy test which returned positive. I had some sort of instinctive feeling that I might be pregnant despite the odds. They then thought I had a miscarriage and sent me home with a packet of Co-codamol, basically I had to just get on with it.
3 days later the second A&E sent me home knowing that I am pregnant with bleeding and abdominal pain but arranged an ultrasound scan with the Early Pregnancy Unit (EPU) 3 days later. A nurse from the EPU phoned me the next day and informed me that my blood test came back and the hCG level was "looking good" meaning that I wasn't miscarrying.
I was aware of my pregnancy for about 6 days and I love my baby from the moment I knew he was there. I stroked my belly and talked to him every day telling him to hang in there and medical help would come soon. He did. I saw his heartbeat on the ultrasound. It really broke my heart when the sonographer said that she had bad news for me and my husband.
I was immediately admitted to a ward and an emergency operation scheduled within 7 hours of the scan. The consultant spoke to me and made it clear that if I do not consent to the procedure that I will eventually die. He said that it now has become an emergency due to the size of the embryo and my tube could rupture anytime. All I could think of in my head was, "my baby is still alive, I saw his heartbeat, he is still hanging on trying to live and trying to grow, he would die if the surgeons remove him!" My baby was around 7 to 7.5 weeks.
I went into surgery at 4pm and woke up at 7pm but was too out of it from the drugs to feel anything for the first 24 hours. I have been secretly shedding tears when I became more lucid. My husband sleeps in a different room after I was discharged from hospital because my dogs love to jump onto the bed and I can't afford to be jumped on after my surgery. My husband now sleeps with the dogs.
I don't cry in front of anyone, I do it when I am alone in bed or in the shower. Like you, babybluebell, this episode represents the end of my dreams. I don't think I can conceive again because I lost one tube and the other tube is affected by scar tissue (the consultant says). I understand exactly how you feel. I am still grieving and crying alone. It is nearly 2 weeks ago since my surgery on 10/24. I found great solace in doing little things to remember my little one (e.g. I bought long burning tea lights and burn one every evening to remember him).
I doubt the emotional pain will ever go away especially if this is your first baby and probably your last. My heart goes out to you. I understand the pain you feel. Lots of hugs to you. Take care of yourself. xxx
Hi my dearest frnd thanks a lot 4 sharing ur trauma i feel much better hoping u all heal i wish we did'nt have this scars cos everytime i c and feel it everything comes back bt GOD WILL HEAL US take care
bumped for feather
This sounds awful and I don't mean it to, but I am so glad to have found this thread. No-one really understands what it is like to have an ectopic. I had a horrible time at the start of 2013 and one which has caused us to write a letter to the hospital because they nearly cost me my life. It's only now, 10 months on, that I can write this and not feel angry, sad, absolutely devastated, confused etc etc. Sorry if this goes on a bit, I think now is the time for me to talk about it openly and not feel overwhelming grief.
Christmas 2012 had all the EP symptoms, bleeding, cramps, shoulder tip pain etc and my GP referred me to EPU who couldn't give me an appointment for 2 1/2 days! When they scanned me (just over 6wks) they told me that I'd either already miscarried or was possibly never pregnant at all! Awful lady. I asked her about my tubes, to which she responded I am the professional and I have done my job properly and checked your tubes! I was immediately referred to a MW who told me that it was doubtful I was ever pregnant, despite 4 positive tests! At this point I should have been sent for bloods, but I wasn't.
This wrong decision changed the rest of my life.
3 weeks later, still bleeding and cramping and feeling dreadful, falling asleep at the drop of a hat all day, I collapsed at 3am at home. Thank goodness i woke my husband up just before, as I was in so much pain. I was rushed to hospital by ambulance for an emergency operation to remove my left ruptured tube and have 3L blood transfusion. The worst part was my husband and daughter were brought in before the operation 'just in case'. I will never ever forget that. I had just over 3L blood in my abdomen and was told that I only had another 2/3 left before I wouldn't have made it. A week later I was discharged and had 6 weeks off.
What helped me was a lot of crying and talking and trying to come to terms with other things in my live. Time really is a healer.
Sadly I suffered a MC at 10 1/2 weeks a few weeks ago and had an ERPC and with support from MN I'm doing ok. I have an extremely supportive GP who has made a huge difference to me and made me feel like all is not lost.
Sorry for a long email, it's been good to put it down 'on paper' what went on at the start of 2013. My advice to everyone, talk and cry x
Hi im doing much more better just wishing that GOD can do a miracle and restore my tubes so that i can conceive again
Hi all just passing through so glad this thread is still active and I'm people are here sharing uplifting and healing!
As I say before when I was on this thread I found great comfort in the fact of not being alone and the pain seemed like it would last forever but I am blessed and dispite the odds I did go on to have my beautiful girl! With hand holding from some of my lovely ladies I got to know through here!
Please keep posting and sharing as it will help the " dealing & healing" process!
Blessings to u all x
Hi everyone. It's sad that we are all here but glad that there is a support network for us.
This is my story- Its been 4 weeks since my ectopic pregnancy surgery. I was pregnancy at 5 and a half weeks with pain and bleeding so went to my GP requesting an early scan. Got a call saying the scan would be in 8 days! I said I wanted it earlier but told it wasn't possible. After a week of pain, calling 111 twice, speaking to different GPs about my symptoms, being repeatedly told that it was a stomach bug- I ended up in A&E. After an excruciating 8 hour wait in A&E I got a scan which showed a rupture in my left tube and internal bleeding. Had surgery straight away to remove the ectopic and my tube, lost 2 units of blood and woke up feeling like my heart had been ripped out.
It is completely devastating and very tough to accept- I went back to work after 2 weeks but finding it incredibly hard. I saw a gynaecologist yesterday who told me the chances of a repeat EP and how the EP has affected my fertility- I found this news devastating, although DH thinks it is positive (he is right- its not all doom and gloom but it is hard for me to see it in a positive light). I was sitting in the maternity ward yesterday surrounded by pregnant women and babies feeling so jealous and sad, which is terrible- I know i have no right to feel like this.
Just seeing who is in the same boat so we can support each other, and any success stories of pregnancy after an EP?
Thank you for listening.
Mimosa - I'm so sorry that you had to endure such terrible treatment and lack of sympathy to how you was feeling!
I'm not sure if you read through this thread but I graduated from having 2 ectopic 1 MMC tubes removed and feeling that I would never hold another child to be sitting here today with my 9 month old girl who is my miracle baby!
The sadness the loss the worry and concerns doesn't go away but it does get easier and you wake up 1 day knowing you have turned a corner!
Hand holding and hugs to you all!
Hi all,
I am new here, feels weird to have registered to Mumsnet now I am not expecting or a mummy but I have spent most of today reading this thread after having a really emotional day yesterday!
My story, I got married on the 5th November, after a quick 2 day honeymoon I arrived back home and wasn't feeling well so on the 8th I took a pregnancy test to find out I was pregnant - my and my husband were over the moon. We had been trying for 8 months but after 12 years on the pill my periods were about 50 days apart and I felt relieved my body could get pregnant!! Had 5 days of walking on cloud 9 and then started to bleed very lightly, relaxed all weekend and my best friend convinced me it was implantation bleeding. After 4 days of bleeding I went to EPU , told too early to scan (hcg 500) & went back 2 days later for bloods, told all positive as bloods doubled (1000) but progesterone low so they decided to try to scan and talk about taking tablets to help pregnancy, felt relieved - 4 hours later rushed to hospital as had unbearable cramps and bleeding and rushed into resus - drip, painkillers etc and it was there that I passed a large tissue (still never been told what it was) confirmed miscarriage and product was taken away to be tested. The whole thing was so traumatic and I howled like I've never heard before when I finally got home. Went for scan following day, told everything passed - ensured miscarriage is so common next time will be fine. Next day had to have bloods again and HCG had gone up yet again so suspected ectopic, hcg kept rising and had an emergency scan 48 hours later - no pregnancy found in womb but consultant said I definitely hadn't miscarried as there could be a normal baby developing or it could be ectopic. Her saying normal pregnancy freaked me out completely. The next day I had another emergency scan which after 30 minutes showed an ectopic in my left tube. Took methotrexate the next day however on Friday 29th I was asked to come in for a second shot as numbers still very high (5500). Went for a scan and was told there was fluid on my stomach but the lady said that was fine I could have the 2nd shot - the head nurse thankfully pops her head in and hears about fluid on the stomach , examines scan and after talking to the consultant I'm told there is no option but to operate as they think tube is rupturing. I was in total shock. 6 hours later I wake up from the operation with my husband and mum by my side, the had removed the pregnancy along with my left tube which was bleeding and rupturing as they were doing the surgery. I had no time to think much about it all. I kind of think I am lucky I did experience what we thought was a miscarriage because we had already started to grieve.
Anyway, 10 days after my operation I am doing well physically and emotionally (today!!) but last week I felt like if been hit by a bus and have cried pretty much every day since the 7th November!! Yesterday I thought I was doing better and feeling positive and then I realised I was sitting with tears streaming down my face and cried for about an hour non stop - then on and off all night. Today I have woken up feeling so much better and now the pain has gone I intend to give myself little tasks / aims every day.
I still have no idea what actually happened when I thought I wad miscarrying, I've been told it is possible I had 2 eggs, 1 I miscarried that was in the womb and the other the ectopic pregnancy. I felt really silly but I had no idea that if it was a miscarriage that it would be so painful - like contractions and that it would look like a large clump of tissue the size of 2 conkers even though I was so early (4.5 weeks and then probably 6 weeks at time of surgery)
I just wanted to share my story so anyone else out there can have a read and might find it helpful - also so I can come on here when I'm not feeling so positive (probably tomorrow , who knows!)
I am so so scared to try again, which won't be for at least 3 months because of the metho shot but mainly I am scared it will happen again.
Anyway a quote a friend send me yesterday that might help others.
"New beginnings are often disguised as painful endings".
hi rhi, mimosa and bushmills, glad that you found this thread and that it is helpful, it was born in a terrible time for me, from a conversation with iloveberries. I'm so glad something good has come of that awful time. and hi again to teary.
teary glad that you are feeling better, people say time does heal and they are right
bushmills and mimosa I'm so shocked at the terrible medical care you received, bush, I'm glad you wrote a complaint, did you get a reply?
rhi congrats on your wedding, though I imagine you've had other things on your mind since then! I love your quote, I think it is very true. I think setting little goals every day is a really good idea. It does help but don't overdo it, I got every bug and lurgy going after my surgery, I got quite run down. Little steps!
To who asked for success stories, I have a wriggly 6 month old DS rolling about on the floor right now. I had a MMC just before the EP, so I really thought I was broken and couldn't have any more children. Don't give up hope just yet.
Big Hug & hand holding been where you are Rhi! Scared of trying again! It's heartbreaking to have such a happy thing turn into pain sadness and heartache!
But hold on to the fact you have a supporting husband that is also dealing with the loss! And you have another tube which means you do have a chance of conceiving a healthy pregnancy/baby!
Hold on to tomorrow's! And your pain will subside not forgotten not erased but won't hurt so much!
As I have said and watching my lil miracle fast asleep in her cot!
Pls I didn't mean to sound patronising in what I said!
Rhi- as I know you've heard it over and over!
I have 3 angels watching over us! And they will always be with me etched on my heart! Footsteps that are in tune with my beating heart!
Tas and Count - thank you for your lovely comforting messages, I can see from this thread what a horrid journey you both went through and it makes my heart happy that you both went on the have gorgeous, healthy babies after such trauma. Today has mainly been a very good day, although during my afternoon nap I did have a nightmare which involved a nurse prodding my stomach and asking me what my scars were and why I've cut my own baby out of my stomach when it was fine, when I woke up I burst out crying and my stomach hurt as I was obviously tensing my muscles through my sleep - I'm hoping this doesn't become a regular thing?!
It is my nature to always see the positives but this is by far the hardest and most painful thing that I've ever gone through. Thankfully my husband and I, although newly married have been together since we were 19 (11 years) so know each other inside out and will grow even stronger because of this I am sure. I have a nice life and generally live in a happy bubble so this experience has really knocked me, a reminder that things like this will come and grab hold of you out of the blue, I intend to hold on for dear life and get through it a stronger person. We will have a healthy baby, I can't think any other way right now. Luckily I have the benefit of volunteering at a hospice every weekend so constantly talk to the patients about grieving, because of this I am not afraid to cry.
Life works in mysterious ways - whilst I have been losing a baby these last 3 weeks, my brother was secretly going through his second session of IVF and making a baby, on Friday they finally got a positive result - 4 years of trying. This should give hope to anyone that has to go down that route. We will be blessed with a baby next year in the family after all.
After the operation I was not expecting such pain! I was walking so bent over I honestly thought they'd pulled all my muscles too tight and that I would never be able to walk standing up straight again!! Annoyingly I got a urine infection after the operation and now the antibiotics have stopped I feel like I am getting thrush!! Anyway hopefully my GP can help me with that.
I am surprised I don't need any bloods taken or checked, I was basically told they won't follow up until I fall pregnant again! I'm worried that because of being on the pill for 11 years and having irregular periods for the 7 months I came off it prior to this happening that they will continue to be even more irregular now this has happened! I was told I had low progesterone when I got my second lot of bloods back - is this what happens with all ectopics or should I be taking something to ensure it isn't low for future pregnancies? Never even knew what progesterone was before this! Pretty sure they didn't go into much detail all those years back at school!
Anyway - hugs to everyone going through this or having been through it , us women really are incredible.
hi rhi I hope you are feeling better now and your GP has given you something for the thrush. I had nightmares after my EP, sounds very normal, and it did pass. Hope that you don't have many more. It sounds like you are letting it all out and are comfortable with grieving, together with a supportive husband, you are very lucky. I know what you mean about life suddenly grabbing you, it reminds you that you never know what is round the corner, so enjoy the good things while they last and be grateful for them! I find I am much more thankful for the good things in life - loving family, good health - I appreciate them much more now. And congrats on becoming an aunty, I hope you find great joy with your niece/nephew.
I think I had a 6 week follow up after my op, just to confirm to me it was an ectopic pg, but they didn't do much, possibly I POAS, but I suppose you could do that at home to confirm they removed everything.
I was advised to keep a close eye on my cycle from then on, so that when I got pg, I could go early and be monitored and scanned. A bit tricky when you're irregular (as I was) but if you know when you ovulate (looking at the EWCM) and you know your luteal phase (same every month) then it's not too bad. My progesterone was low with the EP, I think it's a symptom of the EP rather than a problem with you. When I got pg again it was much higher. Wishing you a speedy recovery and lots of luck when you TTC again x
Hello, hoping someone can answer this. I had surgery for an ectopic pregnancy three days ago. The keyhole surgery to remove foetus and part of the Fallopian tube. I am still very sore on the three scars, which is to be expected.
However, I'm also bleeding vaginally. At first it was dark spotting, now a fair amount of bright red blood. I'm sure this is normal but none of the doctors or nurses mentioned it, only the scars. Is this normal?
And do you know, is it from the surgery (the cuts) or is it effectively the lining of the womb coming away as per a mc?
Thanks in advance.
Hi Hessy, I am sorry to hear you have had to go through this, I hope you are resting well and you have people around you to take care of you. Like you I started having vaginal bleeding 3 days after my operation and it became bright red, heavy and continued for about 4 days, I was told that it is normal although no one explained to me why it happens other than it isn't a period! I would say that unless it gets really heavy or the pain becomes a lot worse rather than better then it is completely fine. In case no one mentioned to you (and I doubt you would want to after your surgery) use pads and not tampons.
I hope you start feeling a bit better soon, if it makes you feel better to know, I was in a lot more pain than I expected the first 4 days but from then on in every day got better pain wise and also emotionally too, although I had a cry pretty much every day for 2 weeks after surgery and this really helped me to deal with it rather than keep it all in.
Anyway I my op was 4 weeks ago tomorrow and I had my pregnancy and left Fallopian tube removed so any support you want or questions feel free to ask!!
Take things really easy and slowly, I did and pretty much lay down or was on the sofa for the entire first week and only did little things the second week, don't rush.
Thinking of you
Hi everyone
I just wanted to add a quick update...
My surgery was just over 6 months ago in June 2013 when I had emergency surgery to remove my right tube which had been badly ruptured by an 8 week ectopic pregnancy. I had internal bleeding too and was very lucky it wasn't even worse as my hcg levels were extremely high, in the 30,000 range.
The most traumatic experience and few weeks of my life.
Anyway after about 6 weeks me and me DH started trying again, every month that went by without a BFP was like a knife to the heart. I felt ovulation pains every month on my 'tubeless side' as I always have felt them on my right side, and practically convinced myself that my left side didn't work at all and I was never going to get pregnant ever again. I got lower each month, cried all the time, started having flashbacks to my surgery and eventually got put on antidepressants at the end of October for 30 days to start with. They did help my mood, a lot, and I was no longer crying every day. We continued ttc as normal and on Nov 21st I got a BFP. I could not believe it. I truly never thought I'd be able to get pregnant again. Then the worry of another ectopic starts to creep in...
I have had two scans, an NHS one at 6 weeks and a private one at 8 weeks. Both confirmed a pregnancy in the right location with a beating heart. I am now 10 weeks and praying everyday that this pregnancy works out, counting the days until my next scan.
I just wanted to share, and again show that there IS hope after an ectopic...and a normal pregnancy afterwards is quite possible. Because I know how it feels to think your fertility is ruined, and I know just how important and encouraging it is to read other people's stories who have been in the same situation.
I hope everyone who (unfortunately) needs to read and use this thread gets all the support that they need and I wish everybody lots of good luck for the future.
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January 12, 2015
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The University of Pardubice and its management headed by the Rector express deep grief over the tragic events and terrorist attacks in Paris on 13 November 2015, offering sympathy to the suffering families and to all relatives of the victims of the insidious attack.
The Rector said, in remembering the 17 November – the Day of the Fight for Freedom and Democracy - on the evening of the 13 November, only a few hours before the tragic events in Paris:
"Today´s world is rather complicated in many aspects, since we face new problems and challenges, even though we live in the most successful period of our history in terms of economic development. We should not trust those who offer seemingly simple solutions. We have to fight against pessimism, negativism, nationalism and intolerance. We must protect the thousand- year-proven traditional values of the society. The danger of this world is presented, among other, by the fact that a substantial part of the society easily assumes the feeling of threat, looking for someone who seems to have clear visions and ability to solve any problem easily.
The academic community is generally responsible for preventing any new totalitarian system from re-gaining its winning position. Let us not forget that freedom and democracy have to be thoroughly protected."
It is important to refrain from thoughtless competition and simple solutions. Democracy and human values are difficult to gain and even more difficult to sustain as it has been reaffirmed by the recent events.
Pardubice, 16 November 2015


text:
Get an overview of this hotelBeaux Arts bed & breakfast in Anacapri with free breakfast
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Popular property highlights
Situated in Anacapri, this bed & breakfast is within a 5-minute walk of Piazza Vittoria and Casa Rossa. Chiesa di San Michele and Chiesa di Santa Sofia are also within 10 minutes.
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This bed & breakfast features free breakfast and free WiFi in public areas.
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All 3 rooms offer comforts like Select Comfort beds and Frette Italian sheets, plus free WiFi and digital TV. Other amenities available to guests include refrigerators, minibars, and room service.Information missing or incorrect? Tell us!Opens in a new window
Cavalluccio Marino
Bed & Breakfast Amenities
Bed & Breakfast Amenities
A complimentary breakfast is offered each morning. Public areas are equipped with complimentary wireless Internet access.
- Total number of rooms - 3
- Number of floors - 1
- Free breakfast
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- Year Built 2010
- Number of buildings/towers - 1
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Room Amenities
- Premium bedding
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A complimentary continental breakfast is served each morning between 6:30 AM and 10:30 AM.
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- Accessible bathroom
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Check-in time starts at 1:00 PM
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Extra-person charges may apply and vary depending on property policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
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Bed & Breakfast Name
- Cavalluccio Marino
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Hilary Duff
Hilary Duff is moving up in the world on entertainment. Hilary Duff and her sister Haylie both are trying to break into the A-List group. Hilary Duff recently announced her engagement to Mike Comrie. Hilary Duff pictures and videos at Gossip Center.
Hilary Duff thinks being divorced "sucks".
The 'Younger' star split from ex-husband Mike Comrie - with whom she shares four-year-old son Luca - last year and admits it was tough at first.
She said: "Some of my friends tell me, 'God, it must be so nice, you get a break from your kid because you share him.' I'm divorced, and it sucks.
"Well, it did suck for a while; now it's just normal. But it's true, I do get a break. I had Luca by myself for a few weeks, no help, when Mike was on the road, and when he got home I was like, 'He's yours! Bye!'"
And the 29-year-old actress and singer isn't too enamoured by the Los Angeles dating scene.
She added: "I never think being single sucks until I go out in Los Angeles with my girlfriends and the options are terrible. I really attract the old men!"
And Hilary admits she can feel guilty about leaving her son behind when she is working.
She added: "I always feel torn or guilty about something. I'm not working right now, and at first I stressed about that, like, 'Oh, I shouldn't stay still this long.' And that's ridiculous.
"I have a child; it's totally fine to not be slammed all the time. It feels really good to not be on someone else's schedule - besides Luca's. I'm basically his chauffeur!"
The blonde beauty hasn't ruled out having any more children in the future.
She told the new issue of Redbook magazine: "I've gotten over the fact that I'm not going to have a baby that's close in age with Luca. It sucked when everyone would be like, 'When are you having another?' I'd be like, 'Do you know what's happening right now? I'm not having another.'
"You get engaged, 'When's the wedding?' You get married, 'When's the baby?' You have a baby, 'When's the next one?' ... I'm cherishing the now. Besides, watch: I'll probably get some evil spawn of Hilary the next time around."
She's a super-hot mom to four-year-old Luca, and Hilary Duff doesn't allow bikini beach season stress her out.
The "Younger" star (28) told Today.com, "I don't care to look absolutely perfect in a bathing suit. I'm a normal girl. I'm strong. I'm fit. I feel good about myself after I've had a baby."
It's been nearly a year since Hilary Duff first filed for divorce from Mike Comrie, and this week the decree has been finalized.
Back in 2014, the "Younger" actress and her hockey-playing beau separated and a year later she submitted her petition for divorce, citing "irreconcilable differences" as the reason for the split.
She's enjoying a career resurgence as of late, but with the upswing in work Hilary Duff has also experienced an increase in inner-conflict.
The "Younger" star, who is also mom to 3-year-old Luca, is featured on the front page of the February 2016 issue of Redbook, and in her interview she confesses, "It's hard to say that you really can have it all. I think you can to a certain degree, but fully loaded, pedal-all-the-way-down is tough. You're missing out on something, either on the career or on the family. When I'm really busy with work, I feel extremely guilty."
Taking some time for herself, Hilary Duff engaged in a spot of shopping in a few top Los Angeles boutiques on Wednesday (August 26).
The "Younger" actress dressed in dark tones, punctuated with a light brown Chanel handbag as she carried her iced coffee beverage up the sidewalk while paparazzi looked on.
Taking care of her to-do list, Hilary Duff hopped in her white Mercedes-Benz SUV and drove into Los Angeles on Tuesday morning (July 21).
The "So Yesterday" songstress, who just released her new music video for "My Kind" last week, wore two different outfits throughout the day, including pink trousers and a black top as well as a pair of jeans and a pullover.
Her life is filled with awesomeness and Hilary Duff gave fans a look at her daily routine in her new music video for "My Kind."
The 27-year-old kicks off the morning with some vocal training followed by a rigorous dance rehearsal and a bottle of Kombucha tea.
Taking care of her to-do list, Hilary Duff ran errands around the Los Angeles area on Thursday afternoon (July 2).
The "Lizzie McGuire" lady recently stated that she's closed her Tinder account- "I'm not still using it. It was really fun for me for a minute; I wanted to experience something totally normal and also shock people. No one thought I was really on it – and I was really on it!"
Following her divorce from Mike Comrie, Hilary Duff has been playing the field with the help of the Tinder iPhone dating app.
Earlier this week, the "Cheaper By the Dozen" dame called in to the Kyle and Jackie O show to play a blind phone date game with eight potential suitors, though the hosts withheld her name.
Capitalizing on her 'single-and-ready-to-mingle' status, Hilary Duff is a dating machine in her new music video for her single "Sparks."
The just-released clip finds the "So Yesterday" songstress getting interviewed on the radio about her Tinder membership as well as chatting with her gal pals about potential dates.
Hottest Videos
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BEST wrap day possible with my very own rain soaked "Tip-Tip" end to an incredible shoot for #Soch..coming soon!! pic.twitter.com/aqNF4Zsji8— Nimrat Kaur (@NimratOfficial) December 6, 2015
And it's a wrap for @AirliftFilm with @akshaykumar @NimratOfficial @nikkhiladvani @RajaMenon Priya Seth, Ahmed Khan! pic.twitter.com/smmLnvKvEn— Vikram Malhotra (@vikramix) December 6, 2015
Wid Akshay Phaji @akshaykumar @AirliftFilm @nikkhiladvani @AmaalMallik
Song shoot pic.twitter.com/kT4GCyTWmE— Kumaar (@kumaarofficial) December 6, 2015
The final song dub. Thank you @TheArijitSingh @AmaalMallik @kumaarofficial. @TSeries @AirliftFilm #Soch pic.twitter.com/CS0DWMY7Us— Nikkhil Advani (@nikkhiladvani) December 1, 2015
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BUENOS AIRES ‒ For many countries in Latin America, demand from China has been essential to maintaining high GDP growth rates over the last decade. But will Chinese demand for commodities be enough to sustain high prices for the region's exports in the coming years?
During the last two decades, four factors combined to generate a sharp increase in world demand for commodities: rapid growth in global GDP, increasing urbanization in developing countries, a rise in population at a rate of 800 million people per decade, and a significant decrease in poverty. With the exception of global population growth, China has been the most dynamic country in all of these respects.
For example, the number of Chinese living in poverty fell by 650 million over the last two decades. Moreover, China accounts for half of the global increase of 1.5 billion people earning between $2-13 a day in the past 20 years.
But should we expect what happened from 1990 to 2010 to continue in the coming decades? To answer that question, several variables must be taken into account: demand growth, technological change, investment, and the commitment to confront global warming, among others. Bearing in mind such complexity, let's consider only some determinants of demand that are linked to increased income.


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Oklahoma and Oklahoma State fans have been wondering what in the world Larry Birdine has been thinking. Birdine finally got a chance to answer some of their questions after the Sooner commitment visited Oklahoma State.
"I went up there so that I would have something to compare my visit to Oklahoma to," Birdine said. "I plan to be in Norman this weekend, and see how it compares to Stillwater."
Will the Oklahoma coaches still have an offer for Birdine?
"I talked to coach (Cale) Gundy last night as a matter of fact," he said. "He said they still definitely want me, and like I say I'll be up there this weekend to make sure that's where I want to be."
Most importantly, is he still committed?
"I wanted to be, but when I talked to the coaches they told me if I wanted to take visits I would have to de-commit," he said. "I am still leaning to Oklahoma and unless I see something this weekend that I really don't like that's where I'll end up."
Birdine said his brother, Oklahoma State alum Jason Gildon, hasn't really been pushing him away from Oklahoma because of the school but said he has some personal differences with the coaches.
"He likes the University and the people and all of that kind of stuff," he said. "He was good friends with Julius McMillan, and Julius got in some trouble for reasons that my brother just didn't agree with. I think that to a point Julius wanted to party too much instead of taking care of some of his business. I'm not really like that, so I'm not too worried about that if I went to Oklahoma."
Either way, Birdine has a tough decision ahead of himself – again.


text:
Definition Edit
An Information operations attack (IO attack)
|may take many forms, for example: (1) to slow adversary computers, the software may be disrupted by transmitting a virus or other malicious code; (2) to disable sophisticated adversary weapons, the computer circuitry may be overheated with directed high energy pulses; and (3) to misdirect enemy sensors, powerful signals may be broadcast to create false images. Other methods for IO attack may include psychological operations such as initiating TV and radio broadcasts to influence the opinions and actions of a target audience, or seizing control of network communications to disrupt an adversary's unity of command.[1]


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